Recombinant host cell for expressing proteins of interest

ABSTRACT

The present invention is in the field of recombinant biotechnology, in particular in the field of protein expression. The invention generally relates to a method of expressing a protein of interest (POI) from a host cell. The invention relates particularly to improving a host cell&#39;s capacity to express and/or secrete a protein of interest and use of the host cell for protein expression. The invention also relates to cell culture technology, and more specifically to culturing cells to produce desired molecules for medical purposes or food products.

FIELD OF INVENTION

The present invention is in the field of recombinant biotechnology, inparticular in the field of protein expression. The invention generallyrelates to a method of expressing a protein of interest (POI) from ahost cell. The invention relates particularly to improving a host cell'scapacity to express and/or secrete a protein of interest and use of thehost cell for protein expression. The invention also relates to cellculture technology, and more specifically to culturing cells to producedesired molecules for medical purposes or food products.

BACKGROUND OF THE INVENTION

Successful production of proteins of interest (POI) has beenaccomplished both with prokaryotic and eukaryotic hosts. The mostprominent examples are bacteria like Escherichia coli, yeasts likeSaccharomyces cerevisiae, Pichia pastoris or Hansenula polymorpha,filamentous fungi like Aspergillus awamori or Trichoderma reesei, ormammalian cells like CHO cells. While the yield of some proteins isreadily achieved at high rates, many other proteins are only produced atcomparatively low levels.

To improve the secretion of a recombinant protein, one strategy is totarget on the host's secretory pathway involving the folding andprocessing of proteins.

Co-expression of a cDNA encoding protein disulfide isomerase (PDI) and acDNA encoding a heterologous disulphide-bonded protein was firstsuggested in WO 93/25676 as a means to increase the yield of theheterologous protein. WO 93/25676 reported that the recombinantexpression of antistasin and tick anticoagulant protein can be increasedby co-expression with PDI.

WO 94/08012 provided methods for increasing the secretion ofoverexpressed protein in yeast by increasing expression of an Hsp70chaperone protein, i.e. KAR2/BiP, or a PDI chaperone protein.

WO 05/0617818 and WO 06/067511 provided methods for producing a desiredheterologous protein in yeast by using a 2 μm-based expression plasmid.It was demonstrated that the yield of a heterologous protein issubstantially increased when the genes for one or more chaperoneprotein(s) and a heterologous protein are co-expressed on the sameplasmid.

WO 2008/128701A2 described an expression system to increase thesecretion of a POI from a eukaryotic cell by employing one of thefollowing proteins BMH2, BFR2, COG6, C0Y1, CUP5, IMH 1, KIN2, SEC31,SSA4 and SSE1.

Another approach to increase protein production is based on theoverexpression of HAC1, a transcription factor that activates theunfolded protein response (UPR). Transcriptional analyses revealed thatmore than 330 genes are regulated by HAC1, most of them being involvedin secretion or in the biogenesis of secretory organelles. WO 01/72783describes methods for increasing the amount of a heterologous proteinsecreted from a eukaryotic cell by inducing an elevated unfolded proteinresponse (UPR), wherein the UPR is modulated by co-expression of aprotein selected from the group consisting of HAC1, PTC2 and IRE1.

Wentz et al. employed a Saccharomyces cerevisiae yeast surface displaygene library to identify improved secretion strains by applying anappropriate selection pressure. The yeast cDNAs CCW12, SED1, CWP2, RPP0were found to enhance the display of scTCR in a temperature-dependentmanner. ERO1 enhanced protein secretion when induced at 20° C. (Wentz etal., Appl. Environ. Microbiol. (2007) 73(4):1189-1198).

Liu et al. (Biotechnol. Prog. (2006), 22:1090-1095) showed thatco-overexpression of Kar2 in Pichia pastoris increases rhG-CSFexpression 5.6 fold. Combining KAR2 with Sec63, PDI and YDJ resulted inan increase of 2.8, 6.5 and 5.94 fold. In the experiments performed byBlatt et al, co-overexpression of KAR2 (BiP) increased A33scFvexpression in Pichia pastoris two-fold. Combining KAR2 with PDI almosteliminated the positive KAR2 effect (Blatt et al., Appl. Microbiol.Biotechnol., 2007, 74:381-389).

Guerfall et al. examined the effect of overexpressing endogenous HAC1 inP. pastoris. Furthermore, HAC1 was overexpressed constitutively andinducibly. In all cases, an increased KAR2 expression as a result ofinduced UPR was identified. Constitutive overexpression of full-lengthHAC1 had little or no effect, while overexpression of the induced formof HAC1 led to an increase of protein secretion in one out of fourcases, and a decrease in three out of four cases (Guerfall et al.,Microbial Cell Factories, (2010), 9:49).

Sleep et al. showed that co-overexpression of Lhs1 increases theconcentration of rHA. LHS1 was combined with SIL1, JEM1 and SCJ1, buttiters were lower than with JEM1 co-overexpressed alone.Co-overexpression of SIL1, LHS1 and JEM1 at the same time increasedGM-CSF expression by 1.45 fold and the rHA expression by approximately1.1 and 2 fold, dependent on the cultivation media (Sleep et al. Appliedand Environmental Microbiology, (2008) 74(24):7759-7766).

US 2009221030 A1 described the co-expression of various helper proteins,inter alia, BiP1, alone and in combination with other helper proteins,inter alia, LHS1, in Trichoderma reesei. The highest expression valueswere obtained with BiP1 alone, while the combination of BiP1 and LHS1lead to 8% lower secreted protein titers.

U.S. Pat. No. 8,440,456 provided the genome sequence of Pichia pastorisand disclosed nucleic acid sequences encoding signal peptides, chaperonsand promoters. It disclosed expression vectors comprising the nucleicacid sequences and genetically engineered yeast capable ofoverexpression of 14 chaperones. ROT1, SHR3 and SIL1 were specificallyselected for testing, however, no expression was observed for SIL1, andoverexpression of ROT1 or SHR3 failed to lead to any significantenhancement of the secretion of heterologous proteins. Whileoverexpression of genes encoding helper proteins is usually applied toenhance expression of a protein of interest, underexpression of a geneencoding a protein or even a knock-out of the gene encoding it is not oronly rarely applied.

High level of protein yield in host cells may be limited at one or moredifferent steps, like folding, disulfide bond formation, glycosylation,transport within the cell, or release from the cell. Many of themechanisms involved are still not fully understood and cannot bepredicted on the basis of the current knowledge of the state-of-the-art,even when the DNA sequence of the entire genome of a host organism isavailable.

There is a constant need for methods to improve a host cell's capacityto produce and/or secret proteins of interest. One object of theinvention is to provide new methods to increase the yield of proteins inhost cells which are simple and efficient and suitable for use inindustrial methods. It is another object to provide host cells toachieve this purpose.

It must be noted that as used herein, the singular forms “a”, “an” and“the” include plural references and vice versa unless the contextclearly indicates otherwise. Thus, for example, a reference to “a hostcell” or “a method” includes one or more of such host cells or methods,respectively, and a reference to “the method” includes equivalent stepsand methods that could be modified or substituted known to those ofordinary skill in the art. Similarly, for example, a reference to“methods” or “host cells” includes “a host cell” or “a method”,respectively.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”. For example, A, B and/or C means A, B, C, A+B, A+C, B+C andA+B+C.

The term “about” or “approximately” as used herein means within 20%,preferably within 10%, and more preferably within 5% of a given value orrange. It includes also the concrete number, e.g., about 20 includes 20.

The term “less than”, “more than” or “larger than” includes the concretenumber. For example, less than 20 means 20 and more than 20 means 20.

Throughout this specification and the claims or items, unless thecontext requires otherwise, the word “comprise” and variations such as“comprises” and “comprising” will be understood to imply the inclusionof a stated integer (or step) or group of integers (or steps). It doesnot exclude any other integer (or step) or group of integers (or steps).When used herein, the term “comprising” can be substituted with“containing”, “composed of”, “including”, “having” or “carrying.” Whenused herein, “consisting of” excludes any integer or step not specifiedin the claim/item. When used herein, “consisting essentially of” doesnot exclude integers or steps that do not materially affect the basicand novel characteristics of the claim/item. In each instance herein anyof the terms “comprising”, “consisting essentially of” and “consistingof” may be replaced with either of the other two terms.

Further, in describing representative embodiments of the presentinvention, the specification may have presented the method and/orprocess of the present invention as a particular sequence of steps.However, to the extent that the method or process does not rely on theparticular order of steps set forth herein, the method or process shouldnot be limited to the particular sequence of steps described. As one ofordinary skill in the art would appreciate, other sequences of steps maybe possible. Therefore, the particular order of the steps set forth inthe specification should not be construed as limitations on the claims.In addition, the claims directed to the method and/or process of thepresent invention should not be limited to the performance of theirsteps in the order written, and one skilled in the art can readilyappreciate that the sequences may be varied and still remain within thespirit and scope of the present invention.

It should be understood that this invention is not limited to theparticular methodology, protocols, material, reagents, and substances,etc., described herein. The terminologies used herein are for thepurpose of describing particular embodiments only and are not intendedto limit the scope of the present invention, which is defined solely bythe claims/items.

All publications and patents cited throughout the text of thisspecification (including all patents, patent applications, scientificpublications, manufacturer's specifications, instructions, etc.),whether supra or infra, are hereby incorporated by reference in theirentirety. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention. To the extent the material incorporated by referencecontradicts or is inconsistent with this specification, thespecification will supersede any such material.

SUMMARY

The present invention is partly based on the surprising findings ofseveral proteins (herein referred to as knockout (KO) proteins),including KO1, KO2, KO3 (also referred to herein as KO protein 1, KOprotein 2, and KO protein 3, respectively) and functional homologuesthereof, whose expression was observed to have a negative impact on theyield of POI from a host cell. The meaning of functional homologue isdefined in the latter part of the application. Furthermore, it has beendiscovered that a modification of the genes such as mutation or deletionis able to increase the yield of POI. This disclosure provides methodsand materials useful for improving the yield of POI by engineering hostcells such that they underexpress the genes identified by the inventors.The term “yield” refers to the amount of POI or model protein(s) asdescribed herein, in particular SDZ-Fab (SEQ ID NO: 25 and 26) andHyHEL-Fab (SEQ ID NO: 29 and 30), respectively, which is, for example,harvested from the engineered host cell, and increased yields can be dueto increased amounts of production or secretion of the POI by the hostcell. Yield may be presented by mg POI/g biomass (measured as dry cellweight or wet cell weight) of a host cell. The term “titer” when usedherein refers similarly to the amount of produced POI or model protein,presented as mg POI/L culture supernatant. An increase in yield can bedetermined when the yield obtained from an engineered host cell iscompared to the yield obtained from a host cell prior to engineering,i.e., from a non-engineered host cell.

Preferably, “yield” when used herein in the context of a model proteinas described herein, is determined as described in Example 5c.Accordingly, the “yield” when used herein in the context of a modelprotein as described herein is also referred to as “Fab yield” or “Fabtiter”. A Fab titer is given as mg/L and a Fab yield as mg/biomass(measured as dry cell weight or wet cell weight).

Briefly, P. pastoris strains CBS7435mut^(s) pPM2d_pAOX HyHEL and/orCBS7435mut^(s) pPM2d_pAOX SDZ (see Example 1 for their generation) whichexpress the model protein HyHEL-Fab and SDZ-Fab, respectively, areengineered with a polynucleotide encoding a helper protein or functionalhomologue thereof as described herein. For co-overexpression, the geneencoding a helper protein is cloned under control of the P. pastoris GAPpromoter and transformed into the Fab producing strains as described inexample 4. For underexpression the gene encoding a KO target or itsfunctional homologue is knocked out from the genome of the Fab producingstrain (see example 6). Engineered cells are grown in YP-mediumcontaining 10 g/L glycerol and 50 μg/mL Zeocin overnight at 25° C. (seeExample 5a). Aliquots of such a culture (corresponding to a final OD₆₀₀of 2.0) are transferred to synthetic medium M2 containing 20 g/L glucoseand a glucose feed tablet (described in Example 5a) and incubated for 25h at 25° C. Cultures are washed and resuspended in synthetic medium M2and aliquots (corresponding to a final OD₆₀₀ of 4.0) are transferredinto synthetic medium M2 supplemented with 5 g/L methanol. Methanol (5g/L) is added 3 more times every 12 hours. After 48 h, cells areharvested by centrifugation. Biomass is determined by measuring theweight of the cell pellet derived from 1 mL cell suspension. Thesupernatant is used for quantification of SDZ-Fab or HyHEL-Fab,respectively, by ELISA (described in Example 5c). Specifically, ananti-human IgG antibody (e.g. ab7497, Abcam) is used as coating antibodyand a e.g. goat anti-human anti-human IgG (Fab specific) antibody (e.g.Sigma A8542, alkaline phosphatase conjugated) is used as detectionantibody. Commercial Human Fab/Kappa, IgG fragment is used as standardwith a starting concentration of 100 ng/mL, supernatant samples arediluted accordingly. An increase in the yield may be determined based ona comparison of POI yield before and after the cell is engineered tounderexpress the polypeptide encoding the KO protein. A standard test asshown in the example may be used to determine the yield difference.

The present invention is partly based on, but not limited to, thefollowing knockout proteins which are discovered by the inventors:

TABLE 1 (Designation Designation in the KO (in analogy to Amino acidExamples (see proteins S. cerevisiae)) sequences Polynucleotides Table7) KO1 FLO8 SEQ ID NO: 10 SEQ ID NO: 22 PP7435_Chr4-0252 KO2 HCH1 SEQ IDNO: 11 SEQ ID NO: 23 PP7435_Chr3-1062 KO3 SCJ1 SEQ ID NO: 12 SEQ ID NO:24 PP7435_Chr1-0176

The present invention provides genetically engineered recombinant hostcells having one or more underexpressed genes encoding the KO protein.As used herein, “engineered” host cells are host cells which have beenmanipulated using genetic engineering, i.e. by human intervention. Whena host cell is “engineered to underexpress” a given protein, the hostcell is manipulated such that the expression level of a functional KOprotein is decreased compared to the host cell under the same conditionprior to the genetic manipulation to achieve underexpression. The hostcell which is to be subjected to genetic engineering comprises at leastone, such as at least 2, or at least 3 polynucleotide sequence encodinga KO protein or functional homologues thereof. Genetic engineeringresults in the underexpression of genes encoding the KO protein.Preferably, the engineering will result in a cell having no detectablelevel for the KO protein(s). Underexpression can be observed bycomparing the engineered host cell with a “corresponding parent strain”which refers to the host cell prior to aforementioned engineering. Asused herein, KO proteins are referred to in the present inventioninterchangeably in plural or singular forms, which however should beunderstood as in singular form unless expressly stated otherwise.

As to the genes that have to be knocked-out—from a chaperone (KO2, KO3)one would have expected that overexpression is beneficial, but it is infact the deletion that has a positive effect.

KO1 which plays a role in flocculation in baker's yeast, one would nothave expected that its deletion enhances protein secretion. Flocculationin baker's yeast is a phenomenon when cells aggregate reversibly in avegetative growth phase, not related to mating.

In a first aspect, the present invention provides a recombinant hostcell for manufacturing a protein of interest, wherein the host cell isengineered to underexpress at least one polynucleotide encoding a KOprotein having an amino acid sequence as shown in SEQ ID NO: 10, 11, 12or a functional homologue thereof, wherein the functional homologue hasat least 30%, such as at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99 or 100%, sequence identity to an amino acid sequenceas shown in SEQ ID NO: 10, 11 or 12.

The recombinant host cell is obtained by first providing a host cellwhich comprises at least one polynucleotide encoding a KO protein havingan amino acid sequence as shown in SEQ ID NO: 10, 11, 12 or a functionalhomologue thereof, wherein the functional homologue has at least 30%,such as at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100%, sequence identity to an amino acid sequence as shown in SEQID NO: 10, 11 or 12, and then engineering the host cell to underexpressthe polynucleotide.

In a preferred embodiment, the host cell is a yeast cell, preferably ahost cell of the genus Pichia and more preferably Pichia pastoris whichdoes not comprise a KO protein of the present invention and/or whichdoes not comprise a polynucleotide encoding the KO protein(s). In oneembodiment, the host cell is one obtained by deleting a polynucleotidesequence as shown in SEQ ID NO: 22, 23, 24, or homologous thereof.

As used herein, a “homologue” or “functional homologue” of a polypeptideshall mean that a polypeptide has the same or conserved residues at acorresponding position in their primary, secondary or tertiarystructure. The term also extends to two or more nucleotide sequencesencoding homologous polypeptides. In particular, polypeptides homologousto the KO1, KO2 and KO3 described in the examples may have at leastabout 30%, such as at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or 100%, amino acid sequence identity with regard to afull-length native sequence or any fragment thereof. Preferably, ahomologous polypeptide will have at least about 35% amino acid sequenceidentity, more preferably at least about 40% amino acid sequenceidentity, more preferably at least about 45% amino acid sequenceidentity, more preferably at least about 50% amino acid sequenceidentity, more preferably at least about 55% amino acid sequenceidentity, more preferably at least about 60% amino acid sequenceidentity, more preferably at least about 65% amino acid sequenceidentity, more preferably at least about 70% amino acid sequenceidentity, more preferably at least about 75% amino acid sequenceidentity, more preferably at least about 80% amino acid sequenceidentity, more preferably at least about 85% amino acid sequenceidentity, more preferably at least about 90%, such as 91, 92, 93, 94,95, 96, 97, 98 or 99% amino acid sequence identity, more preferably atleast about 95% amino acid sequence identity to a native compound, orany other specifically defined fragment of a full-length compound. Whenthe function as a KO1, KO2, and KO3 protein is proven with such ahomologue, the homologue is called “functional homologue”. A functionalhomologue performs the same or substantially the same function as the KOprotein or helper protein from which it is derived from, i.e. itincreases the yield of the model protein SDZ-Fab and/or HyHEL-Fab asdescribed herein. The function can be tested by assay known in the artor preferably with the assay using the model proteins as described inExample 7 or Example 5c, particularly as described herein above in thecontext of “Fab titer” or “Fab yield”.

The function of a potential functional homologue can be tested byproviding expression cassettes into which the functionally equivalentvariant sequences have been inserted, transforming host cells that carrythe sequence encoding a test protein such as one of the model proteinsused in the Example or a specific POI, and determining the difference inthe yield of the model protein or POI under identical conditions.

In one embodiment, the host cell is engineered to underexpress KOprotein 1. The host cell is preferably engineered to underexpress apolynucleotide encoding a protein having an amino acid sequence as shownin SEQ ID NO: 10 or a functional homologue thereof, wherein thefunctional homologue has at least 30%, such as at least 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, sequence identity toan amino acid sequence as shown in SEQ ID NO: 10.

In another embodiment, the host cell is engineered to underexpress KO2.The host cell is preferably engineered to underexpress a polynucleotideencoding a protein having an amino acid sequence as shown in SEQ ID NO:11 or a functional homologue thereof, wherein the functional homologuehas at least 30%, such as at least 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99 or 100%, sequence identity to an amino acidsequence as shown in SEQ ID NO: 11.

In a further embodiment, the host cell is engineered to underexpressKO3. The host cell is preferably engineered to underexpress apolynucleotide encoding a protein having an amino acid sequence as shownin SEQ ID NO: 12 or a functional homologue thereof, wherein thefunctional homologue has at least 30%, such as at least 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, sequence identity toan amino acid sequence as shown in SEQ ID NO: 12.

The present invention provides an isolated polynucleotide sequencecomprising, consisting essentially of, or consisting of SEQ ID NO: 22,SEQ ID NO: 23, or SEQ ID NO: 24, Furthermore, the present inventionprovides an isolated polynucleotide sequence which encodes a polypeptidesequence comprising any one of SEQ ID NO: 10, 11, 12, or functionalhomologues thereof. In some embodiments, the present invention providesan isolated polynucleotide sequence having at least 30%, such as atleast 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or100% sequence identity to a polynucleotide sequence selected from thegroup consisting of SEQ ID NO: 22, 23, 24. More preferably, the isolatedpolynucleotide sequence has 100% sequence identity with SEQ ID NO: 22,SEQ ID NO23, SEQ ID NO: 24.

Furthermore, the present invention provides an isolated polypeptidecomprising the polypeptide sequence of any one of SEQ ID NO: 10, 11, 12,or functional homologues thereof. Preferably, the present inventionprovides an isolated polypeptide comprising the polypeptide sequence ofSEQ ID NO: 10 or functional homologues thereof. Preferably, the presentinvention provides an isolated polypeptide comprising the polypeptidesequence of SEQ ID NO: 11 or functional homologues thereof. Preferably,the present invention provides an isolated polypeptide comprising thepolypeptide sequence of SEQ ID NO: 12 or functional homologues thereof.In a further aspect, the present invention provides an isolatedpolypeptide with a polypeptide sequence having at least 30%, such as atleast 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or100% sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 10, 11, or 12.

The term “nucleotide sequence” or “nucleic acid sequence” used hereinrefers to either DNA or RNA. “Nucleic acid sequence” or “polynucleotidesequence” or simply “polynucleotide” refers to a single ordouble-stranded polymer of deoxyribonucleotide or ribonucleotide basesread from the 5′ to the 3′ end. It includes both self-replicatingplasmids, infectious polymers of DNA or RNA, and non-functional DNA orRNA.

The term “expressing a polynucleotide” means when a polynucleotide istranscribed to mRNA and the mRNA is translated to a polypeptide. Theterm “underexpress” generally refers to any amount less than anexpression level exhibited by a reference standard, which is the hostcell prior to the engineering to underexpress the KO protein. The terms“underexpress,” “underexpressing,” “underexpressed” and“underexpression” in the present invention refer an expression of a geneproduct or a polypeptide at a level less than the expression of the samegene product or polypeptide prior to a genetic alteration of the hostcell or in a comparable host which has not been genetically altered.“Less than” includes, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80, 90%or more. No expression of the gene product or a polypeptide is alsoencompassed by the term “underexpression.”

The host cell may be obtained by providing a cell which expresses KOprotein 1 or functional homologues thereof and engineering the cell tounderexpress the gene which encodes KO protein 1. The host cell may alsobe obtained by providing a cell which expresses KO protein 2 orfunctional homologues thereof and engineering the cell to underexpressthe gene which encodes KO protein 2. Furthermore, the host cell may beobtained by providing a cell which expresses KO protein 3 or functionalhomologues thereof and engineering the cell to underexpress the geneencoding KO protein 3.

In a preferred embodiment, the KO protein 1, when preferablyunderexpressed, may increase the production of the model protein SDZ-Fabor HyHEL-Fab in the host cell by at least 1%, such as at least 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130,140, 150, 160, 170, 180, 190 or at least 200% or more, compared to thehost cell prior to the engineering to underexpress the polynucleotideencoding the KO protein. It has been surprisingly found that exemplaryrecombinant cells described in the Examples were all able to increasethe yield of the model protein SDZ-Fab or HyHEL-Fab by at least 20% (1.2fold change).

In a preferred embodiment, the KO protein 2, when preferablyunderexpressed, may increase the yield of the model protein SDZ-Fab orHyHEL-Fab in the host cell by at least 1%, such as at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190 or at least 200% or more, compared to the hostcell prior to engineering. It has been surprisingly found that exemplaryrecombinant cells described in the Examples were all able to increasethe yield of the model protein SDZ-Fab or HyHEL-Fab by at least 20% (1.2fold change).

In a preferred embodiment, the KO protein 3, when preferablyunderexpressed, may increase the yield of the model protein SDZ-Fab orHyHEL-Fab in the host cell by at least 1%, such as at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190 or at least 200% or more, compared to the hostcell prior to engineering. It has been surprisingly found that exemplaryrecombinant cells described in the Examples were all able to increasethe yield of the model protein SDZ-Fab or HyHEL-Fab by at least 20% (1.2fold change).

In a third aspect, the present invention provides use of any of therecombinant host cells described in the present for manufacturing aprotein of interest.

In a fourth aspect, the present invention provides a method ofincreasing the yield of a protein of interest in a host cell, comprisingunderexpressing at least one polynucleotide encoding a KO protein havingan amino acid sequence as shown in SEQ ID NO: 10, 11, 12 or a functionalhomologue thereof, wherein the functional homologue has at least 30%,such as at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99 or 100%, sequence identity to an amino acid sequence as shown in SEQID NO: 10, 11 or 12.

Preferably, the method comprises underexpressing a polynucleotideencoding a KO protein having an amino acid sequence as shown in SEQ IDNO: 10 or a functional homologue thereof, wherein the functionalhomologue has having at least 30%, such as at least 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, sequence identity to anamino acid sequence as shown in SEQ ID NO: 10.

Preferably, the method comprises underexpressing a polynucleotideencoding a KO protein having an amino acid sequence as shown in SEQ IDNO: 11 or a functional homologue thereof, wherein the functionalhomologue has at least 30%, such as at least 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or 100%, sequence identity to an aminoacid sequence as shown in SEQ ID NO: 11.

Preferably, the method comprises underexpressing a polynucleotideencoding a KO protein having an amino acid sequence as shown in SEQ IDNO: 12 or a functional homologue thereof, wherein the functionalhomologue has at least 30%, such as at least 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99 or 100%, sequence identity to an aminoacid sequence as shown in SEQ ID NO: 12.

In a fifth aspect, the present invention provides a method of increasingthe yield of a protein of interest in a host cell comprising:

-   -   engineering the host cell to underexpress at least one, such as        at least 2, or at least 3, polynucleotide encoding a KO protein        having an amino acid sequence as shown in SEQ ID NO: 10, 11, 12        or a functional homologue thereof, wherein the functional        homologue has at least 30%, such as at least 31, 32, 33, 34, 35,        36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,        52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,        68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,        84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99        or 100%, sequence identity to an amino acid sequence as shown in        SEQ ID NO: 10, 11 or 12,    -   recombining in said host cell a heterologous polynucleotide        encoding a protein of interest, and    -   culturing said host cell under suitable conditions to express        the protein of interest.

In the context of methods for increasing the yield of a protein theorder of the “engineering step” and “recombining step” can alternativelybe reversed such that the “recombining step” precedes the “engineeringstep”. Notably, as described herein, the yield of a protein of interestis increased when a KO protein is underexpressed and/or a helper proteinis overexpressed.

It should be noted that the engineering step and the recombining step donot have to be performed in the recited sequence. It is possible toperform the steps in different order.

Preferably, the present invention provides a method of increasing theyield of a protein of interest in a host cell comprising:

-   -   engineering the host cell to underexpress at least one        polynucleotide encoding a KO protein having an amino acid        sequence as shown in SEQ ID NO: 10, 11, 12 or a functional        homologue thereof, wherein the functional homologue has at least        30%, such as at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,        41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,        57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,        73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,        89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, sequence        identity to an amino acid sequence as shown in SEQ ID NO: 10,    -   recombining in said host cell a heterologous polynucleotide        encoding a protein of interest, and    -   culturing said host cell under suitable conditions to express        the protein of interest.

Preferably, the present invention provides a method of increasing theyield of a protein of interest in a host cell comprising:

-   -   engineering the host cell to underexpress at least one        polynucleotide encoding a KO protein having an amino acid        sequence as shown in SEQ ID NO: 10, 11, 12 or a functional        homologue thereof, wherein the functional homologue has at least        30%, such as at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,        41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,        57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,        73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,        89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, sequence        identity to an amino acid sequence as shown in SEQ ID NO: 11,    -   recombining in said host cell a heterologous polynucleotide        encoding a protein of interest, and    -   culturing said host cell under suitable conditions to express        the protein of interest.

Preferably, the present invention provides a method of increasing theyield of a protein of interest in a host cell comprising:

-   -   engineering the host cell to underexpress at least one        polynucleotide encoding a KO protein having an amino acid        sequence as shown in SEQ ID NO: 10, 11, 12 or a functional        homologue thereof, wherein the functional homologue has at least        30%, such as at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,        41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,        57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,        73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,        89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, sequence        identity to an amino acid sequence as shown in SEQ ID NO: 12,    -   recombining in said host cell a heterologous polynucleotide        encoding a protein of interest, and    -   culturing said host cell under suitable conditions to express        the protein of interest.

In a sixth aspect, the present invention provides a method of producinga protein of interest in host cell comprising:

-   -   providing a host cell engineered to underexpress at least one,        such as at least 2, or at least 3 polynucleotide encoding a KO        protein sequence as shown in SEQ ID NO: 10, 11, 12 or a        functional homologue thereof, wherein the functional homologue        has an amino acid having at least 30%, such as at least 31, 32,        33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,        49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,        65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,        81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,        97, 98, 99 or 100%, sequence identity to an amino acid sequence        as shown in SEQ ID NO: 10, 11 or 12, wherein said host cell        comprises a heterologous polynucleotide encoding a protein of        interest; and    -   culturing the host cell under suitable conditions to express the        protein of interest.

Preferably, the present invention provides a method of producing aprotein of interest in a host cell comprising:

-   -   providing a host cell engineered to underexpress at least one        polynucleotide encoding a KO protein having an amino acid        sequence as shown in SEQ ID NO: 10, 11, 12 or a functional        homologue thereof, wherein the functional homologue has at least        30%, such as at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,        41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,        57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,        73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,        89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, sequence        identity to an amino acid sequence as shown in SEQ ID NO: 10,        wherein said host cell comprises a heterologous polynucleotide        encoding a protein of interest; and    -   culturing the host cell under suitable conditions to express the        protein of interest.

Preferably, the present invention provides a method of producing aprotein of interest in a host cell comprising:

-   -   providing a host cell engineered to underexpress at least one        polynucleotide encoding a KO protein having an amino acid        sequence as shown in SEQ ID NO: 10, 11, 12 or a functional        homologue thereof, wherein the functional homologue has at least        30%, such as at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,        41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,        57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,        73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,        89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, sequence        identity to an amino acid sequence as shown in SEQ ID NO: 11,        wherein said host cell comprises a heterologous polynucleotide        encoding a protein of interest; and    -   culturing the host cell under suitable conditions to express the        protein of interest.

Preferably, the present invention provides a method of producing aprotein of interest in host cell comprising:

-   -   providing a host cell engineered to underexpress at least one        polynucleotide encoding a KO protein having an amino acid        sequence as shown in SEQ ID NO: 10, 11, 12 or a functional        homologue thereof, wherein the functional homologue has at least        30%, such as at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,        41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,        57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,        73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,        89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, sequence        identity to an amino acid sequence as shown in SEQ ID NO: 12,        wherein said host cell comprises a heterologous polynucleotide        encoding a protein of interest; and    -   culturing the host cell under suitable conditions to express the        protein of interest.

The present invention encompasses the underexpression of 2 or all ofKO1, KO2 and KO3 genes. The present invention also encompasses therecombinant host cell for manufacturing a protein of interest, whereinthe host cell is engineered to underexpress at least 2, 3 or morepolynucleotide encoding a KO protein having an amino acid sequence asshown in SEQ ID NO: 10, 11, 12 or a functional homologue thereof,wherein the functional homologue has at least 30% sequence identity toan amino acid sequence as shown in SEQ ID NO: 10, 11 or 12. In someembodiments 2, 3, 4, 5, 6 or more of KO proteins selected from SEQ IDNO: 10, 11, 12 or functional homologues thereof are underexpressed inthe host cell. A functional homologue may be a biologically activefragment of the KO proteins. Generally, biologically active fragment ofa protein shall mean a fragment that exerts a biological effect similaror comparable to that of the full length protein. Such fragments orvariants can be produced e.g. by amino- and/or carboxy-terminaldeletions as well as by internal deletions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid and polynucleotide sequences of the HP1,HP2, HP 3, HP 4, HP5, HP6, HP7, HP8, HP9, HP10, KO1, KO2 and KO3.

FIG. 2 shows the amino acid and polynucleotide sequences of the heavychain and light chain of the model proteins SDZ-Fab and HyHEL-Fab,respectively.

ITEMS OF THE INVENTION

-   -   1. A recombinant host cell for manufacturing a protein of        interest, wherein the host cell is engineered to underexpress at        least one polynucleotide encoding a KO protein having an amino        acid sequence as shown in SEQ ID NO: 10, 11, 12 or a functional        homologue thereof, wherein the functional homologue has at least        30% sequence identity to an amino acid sequence as shown in SEQ        ID NO: 10, 11 or 12.    -   2. The host cell of item 1, wherein said polynucleotide encoding        the KO protein or a functional homologue thereof, preferably        when underexpressed, increases the yield of the model protein        SDZ-Fab (SEQ ID NO: 25 and 26) and/or HyHEL-Fab (SEQ ID NO: 29        and 30), preferably by at least 20% compared to the host cell        prior to engineering.    -   3. The host cell in item 1 or 2, wherein underexpression is        achieved by knocking out the polynucleotide encoding the KO        protein or a functional homologue thereof from the genome of        said the host cell.    -   4. The host cell in item 1 or 2, wherein underexpression is        achieved by disrupting the polynucleotide encoding the KO        protein or a functional homologue thereof in the host cell.    -   5. The host cell in item 1 or 2, wherein underexpression is        achieved by disrupting a promoter which is operably linked with        said polypeptide encoding the KO protein or a functional        homologue thereof.    -   6. The host cell in item 1 or 2, wherein underexpression is        achieved by post-transcriptional gene silencing.    -   7. The host cell in any one of the preceding items, wherein the        host cell is a Pichia pastoris, Hansenula polymorpha,        Trichoderma reesei, Saccharomyces cerevisiae, Kluyveromyces        lactis, Yarrowia lipolytica, Pichia methanolica, Candida        boidinii, and Komagataella, and Schizosaccharomyces pombe.    -   8. The host cell of any of the preceding items, wherein the        protein of interest is an enzyme, a therapeutic protein, a food        additive or feed additive, preferably a detoxifying enzyme.    -   9. The host cell of item 8, wherein the therapeutic protein        comprises an antibody or antibody fragment    -   10. The host cell in any of the preceding items, wherein said        polynucleotide encoding the KO protein, preferably when        underexpressed, increases the yield of the model protein SDZ-Fab        (SEQ ID NO: 25 and 26) and/or HyHEL-Fab (SEQ ID NO: 29 and 30)        by preferably at least 20% compared to the host cell prior to        engineering.    -   11. The host cell in any one of the preceding items, wherein the        host cell underexpresses said at least one polynucleotide        encoding the KO protein or a functional homologue thereof and        expresses the protein of interest.    -   12. The host cell in any one of the preceding items, wherein the        host cell comprises at least one polynucleotide encoding a        helper protein.    -   13. The host cell of item 12, wherein the helper protein has an        amino acid sequence as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,        8, 9, or 162 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,        8, 9, or 162.    -   14. The host cell of item 12 or 13, wherein the host cell is        engineered to express or overexpress the helper protein or a        functional homologue thereof.    -   15. The host cell of any one of the preceding claims, wherein        the host cell is engineered to        -   (i) underexpress a polynucleotide encoding a protein having            an amino acid as shown in SEQ ID NO: 10 or a functional            homologue thereof and further engineered to overexpress a            polynucleotide encoding a helper protein having an amino            acid sequence as shown in SEQ ID NO: 1 or a functional            homologue thereof and a helper protein having an amino acid            sequence as shown in SEQ ID NO: 2 or a functional homologue            thereof, or        -   (ii) underexpress a polynucleotide encoding a protein having            an amino acid as shown in SEQ ID NO: 10 or a functional            homologue thereof and further engineered to overexpress a            polynucleotide encoding a helper protein having an amino            acid sequence as shown in SEQ ID NO: 4 or a functional            homologue thereof and SEQ ID NO: 1 or a functional homologue            thereof.    -   16. A host cell obtained by providing a cell which expresses KO        protein 1 or functional homologues thereof and engineering the        cell to underexpress the gene which encodes KO1.    -   17. A host cell obtained by providing a cell which expresses KO        protein 2 or functional homologues thereof and engineering the        cell to underexpress the gene which encodes KO2.    -   18. A host cell obtained by providing a cell which expresses KO        protein 3 or functional homologues thereof and engineering the        cell to underexpress the gene encoding KO3.    -   19. Use of the host cell in any one of the preceding items for        manufacturing a protein of interest.    -   20. A method of increasing the yield of a protein of interest in        a host cell, comprising underexpressing at least one        polynucleotide encoding a KO protein having an amino acid        sequence as shown in SEQ ID NO: 10, 11, 12 or a functional        homologue thereof, wherein the functional homologue has at least        30% sequence identity to an amino acid sequence as shown in SEQ        ID NO: 10, 11 or 12.    -   21. A method of increasing the yield of a protein of interest in        a host cell comprising:        -   engineering the host cell to underexpress at least one            polynucleotide encoding a KO protein having an amino acid            sequence as shown in SEQ ID NO: 10, 11, 12 or a functional            homologue thereof, wherein the functional homologue has at            least 30% sequence identity to an amino acid sequence as            shown in SEQ ID NO: 10, 11 or 12,        -   recombining in said host cell a heterologous polynucleotide            encoding a protein of interest,        -   culturing said host cell under suitable conditions to            express the protein of interest, and optionally        -   isolating the protein of interest from the cell culture.    -   22. A method of producing a protein of interest in a host cell        comprising:        -   providing a host cell engineered to underexpress at least            one polynucleotide encoding an KO protein having an amino            acid sequence as shown in SEQ ID NO: 10, 11, 12 or a            functional homologue thereof, wherein the functional            homologue has at least 30% sequence identity to an amino            acid sequence as shown in SEQ ID NO: 10, 11 or 12, wherein            said host cell comprises a heterologous polynucleotide            encoding a protein of interest;        -   culturing the host cell under suitable conditions to express            protein of interest, and optionally        -   isolating the protein of interest from the cell culture.    -   23. The method in any one of items 20-22, wherein        underexpression is achieved by knocking out the polynucleotide        encoding the KO protein or a functional homologue thereof from        the genome of said the host cell.    -   24. The method in any one of items 20-22, wherein        underexpression is achieved by disrupting the polynucleotide        encoding the KO protein or a functional homologue thereof in the        host cell.    -   25. The method in any one of items 20-23, wherein        underexpression is achieved by disrupting a promoter which is        operably linked with said polypeptide encoding the KO protein or        a functional homologue thereof.    -   26. The method in any one of items 20-24, wherein        underexpression is achieved by post-transcriptional gene        silencing, preferably by expression of a heterologous RNA        sequence which binds to transcripts from the polynucleotide        encoding the KO protein.    -   27. The method in any one of items 20-26, wherein the host cell        is a Pichia pastoris, Hansenula polymorpha, Trichoderma reesei,        Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia        lipolytica, Pichia methanolica, Candida boidinii, and        Komagataella, and Schizosaccharomyces pombe.    -   28. The method in any of items 20-27, wherein the protein of        interest is an enzyme, a therapeutic protein, a food additive or        feed additive, preferably a detoxifying enzyme.    -   29. The method of item 28, wherein the therapeutic protein        comprises an antibody, or antibody fragment.    -   30. The method of any of items 20-29, wherein said        polynucleotide encoding the KO protein, preferably when        underexpressed, increases the yield of the model protein SDZ-Fab        (SEQ ID NO: 25 and 26) and/or HyHEL-Fab (SEQ ID NO: 29 and 30)        by preferably at least 20% compared to the host cell prior to        engineering    -   31. The method in any one of items 20-30, wherein the host cell        comprises a polynucleotide encoding at least one helper protein.    -   32. The method of item 31, wherein the helper protein has an        amino acid sequence as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,        8, 9, or 162 or a functional homologue thereof, wherein the        functional homologue has at least 30% sequence identity to an        amino acid sequence as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7,        8, 9, or 162.    -   33. The method of any one of items 31 or 32, wherein the host        cell is engineered to express or overexpress a helper protein        having an amino acid sequence as shown in SEQ ID NO: 1, 2, 3, 4,        5, 6, 7, 8, 9, or 162 or a functional homologue thereof, wherein        the functional homologue has at least 30% sequence identity to        an amino acid sequence as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6,        7, 8, 9, or 162.    -   34. A composition comprising at least 10%, 20%, 30%, 40%, or 50%        of a protein of interest and a polynucleotide encoding a helper        protein as defined in item 13, wherein said polynucleotide is        operably linked with a heterologous promoter.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is concerned with the manipulation of KO1, KO2and/or KO3 genes in host cells. It has been surprisingly found that themanipulated host has an increased capacity to produce an expressedprotein of interest.

In a first aspect, the preset invention provides a recombinant host cellfor manufacturing a protein of interest, wherein the host cell isengineered to underexpress at least one, such as at least 2, or at least3, polynucleotide encoding a KO protein having an amino acid sequence asshown in SEQ ID NO: 10, 11, 12 or a functional homologue thereof,wherein the functional homologue has at least 30% sequence identity toan amino acid sequence as shown in SEQ ID NO: 10, 11 or 12.

For the purpose of the present invention the term “KO protein” is alsomeant to encompass functional homologues of KO1, KO2, or KO3.

KO Proteins 1

The function of FLO8 in baker's yeast is presumably a transcriptionalactivator that is required for diploid filamentous growth and haploidinvasive growth and flocculation. It has never been suggested to lead toan increased yield of protein production in a yeast or other host cell.

KO Protein 2

KO2 encodes in baker's yeast the co-chaperone HCH1. HCH1 in some yeastswere found to regulate Hsp90 function (Armstrong et al., PLoS One. 2012;7(11):e49322.). It has never been suggested before that theunderexpression of this protein leads to an increased yield of POI in ahost cell.

KO Protein 3

KO protein 3 encodes in baker's yeast one of several homologs of thebacterial chaperone DnaJ. It has never been suggested before that theunderexpression of this protein leads to an increased yield of POI inthe host cell.

From a chaperone (KO2, KO3) one would have expected that overexpressionis beneficial, but it is in fact the deletion that has a positiveeffect. For KO1 which plays a role in flocculation in baker's yeast, onewould not have expected that its deletion enhances protein secretion.Flocculation in baker's yeast is a phenomenon when cells aggregatereversibly in a vegetative growth phase, not related to mating. However,a skilled person would most likely not have assumed that knocking-out aflocculation gene enhances protein secretion.

“Sequence identity” or “% identity” refers to the percentage of residuematches between at least two polypeptide or polynucleotide sequencesaligned using a standardized algorithm. Such an algorithm may insert, ina standardized and reproducible way, gaps in the sequences beingcompared in order to optimize alignment between two sequences, andtherefore achieve a more meaningful comparison of the two sequences. Forpurposes of the present invention, the sequence identity between twoamino acid sequences or nucleotide is determined using the NCBI BLASTprogram version 2.2.29 (Jan. 6, 2014) (Altschul et al., Nucleic AcidsRes. (1997) 25:3389-3402). Sequence identity of two amino acid sequencescan be determined with blastp set at the following parameters: Matrix:BLOSUM62, Word Size: 3; Expect value: 10; Gap cost: Existence=11,Extension=1; Filter=low complexity activated; Filter String: L;Compositional adjustments: Conditional compositional score matrixadjustment. For purposes of the present invention, the sequence identitybetween two nucleotide sequences is determined using the NCBI BLASTprogram version 2.2.29 (Jan. 6, 2014) with blastn set at the followingparameters: Word Size: 11; Expect value: 10; Gap costs: Existence=5,Extension=2; Filter=low complexity activated; Match/Mismatch Scores: 2,-3; Filter String: L; m.

Host Cell

As used herein, a “host cell” refers to a cell which is capable ofprotein expression and optionally protein secretion. Such host cell isapplied in the methods of the present invention. For that purpose, forthe host cell to express a polypeptide, a nucleotide sequence encodingthe polypeptide is present or introduced in the cell. Host cellsprovided by the present invention can be prokaryotes or eukaryotes. Aswill be appreciated by one of skill in the art, a prokaryotic cell lacksa membrane-bound nucleus, while a eukaryotic cell has a membrane-boundnucleus. Examples of eukaryotic cells include, but are not limited to,vertebrate cells, mammalian cells, human cells, animal cells,invertebrate cells, plant cells, nematodal cells, insect cells, stemcells, fungal cells or yeast cells.

Examples of yeast cells include but are not limited to the Saccharomycesgenus (e.g. Saccharomyces cerevisiae, Saccharomyces kluyveri,Saccharomyces uvarum), the Komagataella genus (Komagataella pastoris,Komagataella pseudopastoris or Komagataella phaffii), Kluyveromycesgenus (e.g. Kluyveromyces lactis, Kluyveromyces mandanus), the Candidagenus (e.g. Candida utifis, Candida cacaoi, the Geotrichum genus (e.g.Geotrichum fermentans), as well as Hansenula polymorpha and Yarrowiafipolytica.

The genus Pichia is of particular interest. Pichia comprises a number ofspecies, including the species Pichia pastoris, Pichia methanolica,Pichia kluyveri, and Pichia angusta. Most preferred is the speciesPichia pastoris.

The former species Pichia pastoris has been divided and renamed toKomagataella pastoris and Komagataella phaffii. Therefore Pichiapastoris is synonymous for both Komagataella pastoris and Komagataellaphaffii.

Examples for Pichia pastoris strains useful in the present invention areX33 and its subtypes GS115, KM71, KM71H; CB57435 (mut+) and its subtypesCB57435 mut^(s), CBS7435 mut^(s)□ΔArg, CBS7435 mut^(s)□ΔHis, CBS7435mut^(s)□ΔArg, ΔHis, CBS7435 mut^(s) PDI⁺, CBS 704 (=NRRL Y-1603=DSMZ70382), CBS 2612 (=NRRL Y-7556), CBS 9173-9189 and DSMZ 70877 as well asmutants thereof.

Examples of E. coli include those derived from Escherichia coli K12strain, specifically, HMS 174, HMS174 (DE3), NM533, XL1-Blue, C600, DH1,HB101, JM109, as well as those derived from B-strains, specificallyBL-21, BL21 (DE3) and the like.

According a further preferred embodiment, the host cell is a Pichiapastoris, Hansenula polymorpha, Trichoderma reesei, Saccharomycescerevisiae, Kluyveromyces lactis, Yarrowia lipolytica, Pichiamethanolica, Candida boidinii, and Komagataella, and Schizosaccharomycespombe. It may also be a host cell from Ustilago maydis.

Preferably, the helper proteins expressed by the host cell is from thesame cell or recombined from a cell of the same species, genus orfamily. As used herein, “recombinant” refers to the alteration ofgenetic material by human intervention. Typically, recombinant refers tothe manipulation of DNA or RNA in a virus, cell, plasmid or vector bymolecular biology (recombinant DNA technology) methods, includingcloning and recombination. A recombinant cell, polypeptide, or nucleicacid can be typically described with reference to how it differs from anaturally occurring counterpart (the “wild-type”). A “recombinant cell”or “recombinant host cell” refers to a cell or host cell that has beengenetically altered to comprise a nucleic acid sequence which was notnative to said cell.

The term “manufacture” or “manufacturing” as used presently refers tothe process in which the protein of interest is expressed. A “host cellfor manufacturing a protein of interest” refers to a host cell in whichnucleic acid sequences encoding a protein of interest may be introduced.The recombinant host cell within the present invention does notnecessarily contain the nucleic acid sequences encoding a protein ofinterest. It is appreciated by a skilled person in the art that the hostcells can be provided for inserting desired nucleotide sequences intothe host cell, for example, in a kit.

The terms “polypeptide” and “protein” are interchangeably used. The term“polypeptide” refers to a protein or peptide that contains two or moreamino acids, typically at least 3, preferably at least 20, morepreferred at least 30, such as at least 50 amino acids. Accordingly, apolypeptide comprises an amino acid sequence, and, thus, sometimes apolypeptide comprising an amino acid sequence is referred to herein as a“polypeptide comprising a polypeptide sequence”. Thus, herein the term“polypeptide sequence” is interchangeably used with the term “amino acidsequence”.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that function in amanner similar to a naturally occurring amino acid.

In a preferred embodiment, the present invention provides a host cellengineered to underexpress the polynucleotide encoding a KO proteinhaving an amino acid sequence as shown in SEQ ID NO: 10, 11, 12 or afunctional homologue thereof, wherein the functional homologue has atleast 30% sequence identity to an amino acid sequence as shown in SEQ IDNO: 10, 11 or 12, wherein when the underexpression takes place, theyield of the model protein SDZ-Fab (SEQ ID NO: 25 for heavy chain andSEQ ID NO: 26 for light chain; FIG. 2) and/or HyHEL-Fab (SEQ ID NO: 29for heavy chain and SEQ ID NO: 30 for light chain; FIG. 2) is increasedby at least 10% compared to the host cell prior to engineering.

As used herein, “engineered” host cells are host cells which have beenmanipulated using genetic engineering, i.e. by human intervention. Whena host cell is “engineered to underexpress” a given protein, the hostcell is manipulated such that the host cell has no longer the capabilityto express a KO protein or functional homologue such as a non-engineeredhost cell with, thereby expression of a given protein, e.g. POI or modelprotein is increased compared to the host cell under the same conditionprior to manipulation.

“Prior to engineering” when used in the context of host cells of thepresent invention means that such host cells are not engineered suchthat a polynucleotide encoding a KO protein or functional homologuethereof is underexpressed. Said term thus also means that host cells donot underexpress a polynucleotide encoding a KO protein or functionalhomologue thereof or are not engineered to underexpress a polynucleotideencoding a KO protein or functional homologue thereof.

The term “underexpression” includes any method that prevents or reducesthe functional expression of one or more of KO1, KO2, KO3 or functionalhomologues thereof. This results in the incapability or reduction toexert its known function. Means of underexpression may include genesilencing (e.g. RNAi genes antisense), knocking-out, altering expressionlevel, altering expression pattern, by mutagenizing the gene sequence,disrupting the sequence, insertions, additions, mutations, modifyingexpression control sequences, and the like. As mentioned herein, a hostcell of the present invention is preferably engineered to underexpress apolynucleotide encoding a protein having an amino acid as definedherein. This includes that, if a host cell may have more than one copyof such a polynucleotide, also the other copies of such a polynucleotideare underexpressed. For example, a host cell of the present inventionmay not only be haploid, but it may be diploid, tetraploid or even more-ploid. Accordingly, in a preferred embodiment all copies of such apolynucleotide are underexpressed, such as two, three, four, five, sixor even more copies.

Method of Underexpression

Preferably, underexpression is achieved by knocking-out thepolynucleotide encoding the KO protein in the host cell. A gene can beknocked out by deleting the entire or partial coding sequence. Methodsof making gene knockouts are known in the art, e.g., see Kuhn and Wurst(Eds.) Gene Knockout Protocols (Methods in Molecular Biology) HumanaPress (Mar. 27, 2009). A gene can also be knocked out by removing partor all of the gene sequence. Alternatively, a gene can be knocked-out orinactivated by the insertion of a nucleotide sequence, such as aresistance gene. Alternatively, a gene can be knocked-out or inactivatedby inactivating its promoter.

In an embodiment, underexpression is achieved by disrupting thepolynucleotide encoding the gene in the host cell.

A “disruption” is a change in a nucleotide or amino acid sequence, whichresulted in the addition, deleting, or substitution of one or morenucleotides or amino acid residues, as compared to the original sequenceprior to the disruption.

An “insertion” or “addition” is a change in a nucleic acid or amino acidsequence in which one or more nucleotides or amino acid residues havebeen added as compared to the original sequence prior to the disruption.

A “deletion” is defined as a change in either nucleotide or amino acidsequence in which one or more nucleotides or amino acid residues,respectively, have been removed (i.e., are absent). A deletionencompasses deletion of the entire sequence, deletion of part of thecoding sequence, or deletion of single nucleotides or amino acidresidues.

A “substitution” generally refers to replacement of nucleotides or aminoacid residues with other nucleotides or amino acid residues.“Substitution” can be performed by site-directed mutation, generation ofrandom mutations, and gapped-duplex approaches (See e.g., U.S. Pat. No.4,760,025; Moring et al., Biotech. (1984) 2:646; and Kramer et al.,Nucleic Acids Res., (1984) 12:9441). Site-directed mutagenesis can beaccomplished in vitro by PCR involving the use of oligonucleotideprimers containing the desired mutation. Site-directed mutagenesis canalso be performed in vitro by cassette mutagenesis involving thecleavage by a restriction enzyme at a site in the plasmid comprising apolynucleotide encoding the parent and subsequent ligation of anoligonucleotide containing the mutation in the polynucleotide. Usuallythe restriction enzyme that digests the plasmid and the oligonucleotideis the same, permitting sticky ends of the plasmid and the insert toligate to one another. See, e.g., Scherer and Davis, 1979, Proc. Natl.Acad. Sci. USA 76: 4949-4955; and Barton et ai, 1990, Nucleic Acids Res.18: 7349-4966. Site-directed mutagenesis can also be accomplished invivo by methods known in the art. See, e.g., U.S. Patent ApplicationPublication No. 2004/0171 154; Storici et ai, 2001, Nature Biotechnol.19: 773-776; Kren et ai, 1998, Nat. Med. 4: 285-290; and Calissano andMacino, 1996, Fungal Genet. Newslett. 43: 15-16. Synthetic geneconstruction entails in vitro synthesis of a designed polynucleotidemolecule to encode a polypeptide of interest. Gene synthesis can beperformed utilizing a number of techniques, such as the multiplexmicrochip-based technology described by Tian et al. (2004, Nature 432:1050-1054) and similar technologies wherein oligonucleotides aresynthesized and assembled upon photo-programmable microfluidic chips.Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be usedinclude error-prone PCR, phage display (e.g., Lowman et al, 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204) andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7:127). Mutagenesis/shuffling methods can be combinedwith high-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides expressed by host cells (Ness et a/.,1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules thatencode active polypeptides can be recovered from the host cells andrapidly sequenced using standard methods in the art. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide. Semisynthetic gene construction isaccomplished by combining aspects of synthetic gene construction, and/orsite-directed mutagenesis, and/or random mutagenesis, and/or shuffling.Semisynthetic construction is typified by a process utilizingpolynucleotide fragments that are synthesized, in combination with PCRtechniques. Defined regions of genes may thus be synthesized de novo,while other regions may be amplified using site-specific mutagenicprimers, while yet other regions may be subjected to error-prone PCR ornon-error prone PCR amplification. Polynucleotide subsequences may thenbe shuffled. Alternatively, homologues can be obtained from a naturalsource such as by screening cDNA libraries of closely or distantlyrelated microorganisms.

Preferably, disruption results in a frame shift mutation, early stopcodon, point mutations of critical residues, translation of a nonsenseor otherwise non-functional protein product.

In another embodiment, underexpression is achieved by disrupting thepromoter which is operably linked with said polypeptide encoding the KOprotein. A promoter directs the transcription of a downstream gene. Thepromoter is necessary, together with other expression control sequencessuch as ribosomal binding sites, transcriptional start and stopsequences, translational start and stop sequences, and enhancer oractivator sequences, to express a given gene. Therefore, it is alsopossible to disrupt any of the expression control sequence to hinder theexpression of the polypeptide encoding the KO protein.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence on the same nucleic acidmolecule. For example, a promoter is operably linked with a codingsequence of a recombinant gene when it is capable of effecting theexpression of that coding sequence.

In another embodiment, underexpression is achieved bypost-transcriptional gene silencing (PTGS). A technique commonly used inthe art, PTGS reduces the expression level of a gene via expression of aheterologous RNA sequence, frequently antisense to the gene requiringdisruption (Lechtreck et al., J. Cell Sci (2002). 115:1511-1522; Smithet al., Nature (2000). 407:319-320; Furhmann et al., J. Cell Sci (2001).114:3857-3863; Rohr et al., Plant J (2004). 40(4):611-21.Post-transcriptional gene silencing is a biological process in which RNAmolecules inhibit gene expression, typically by causing the destructionof specific mRNA molecules using small RNAs including microRNA (miRNA),small interfering RNA (siRNA) siRNA, or antisense RNA. Gene silencingcan occur either through the blocking of transcription (in the case ofgene-binding), the degradation of the mRNA transcript (e.g. by smallinterfering RNA (siRNA) or RNase-H dependent antisense), or through theblocking of either mRNA translation, pre-mRNA splicing sites, ornuclease cleavage sites used for maturation of other functional RNAs,including miRNA (e.g. by Morpholino oligos or other RNase-H independentantisense). These small RNAs can bind to other specific messenger RNA(mRNA) molecules and decrease their activity, for example by preventingan mRNA from producing a protein. Exemplary siRNA molecules have alength from about 10-50 or more nucleotides. The small RNA moleculescomprise at least one strand that has a sequence that is “sufficientlycomplementary” to a target mRNA sequence to direct target-specific RNAinterference (RNAi). Small interfering RNAs can originate from insidethe cell or can be exogenously introduced into the cell. Once introducedinto the cell, exogenous siRNAs are processed by the RNA-inducedsilencing complex (RISC). The siRNA is complementary to the target mRNAto be silenced, and the RISC uses the siRNA as a template for locatingthe target mRNA. After the RISC localizes to the target mRNA, the RNAcan be cleaved by a ribonuclease. The strand has a sequence sufficientto trigger the destruction of the target mRNA by the RNAi machinery orprocess is commonly referred to as an antisense strand in the context ofa ds-siRNA molecule. The siRNA molecule can be designed such that everyresidue is complementary to a residue in the target molecule. PTGS isfound in many organisms. For yeast cells, the fission yeast,Schizosaccharomyces pombe, has an active RNAi pathway involved inheterochromatin formation and centromeric silencing (Raponi et al.,Nucl. Acids Res. (2003) 31(15): 4481-4489). Some budding yeasts,including Saccharomyces cerevisiae, Candida albicans and Kluyveromycespolysporus were also found to have such RNAi pathway (Bartel et la.,Science Express doi:10.1126/science.1176945, published online 10 Sep.2009). “Underexpression” can be achieved with any known techniques inthe art which lowers gene expression. For example, the promoter which isoperably linked with the polypeptide encoding the KO protein can bereplaced with another promoter which has lower promoter activity.Promoter activity may be assessed by its transcriptional efficiency.This may be determined directly by measurement of the amount of mRNAtranscription from the promoter, e.g. by Northern Blotting, quantitativePCR or indirectly by measurement of the amount of gene product expressedfrom the promoter.

Underexpression may in another embodiment be achieved by intervening inthe folding of the expressed KO protein so that the KO protein is notproperly folded to become functional. For example, mutation can beintroduced to remove a disulfide bond formation of the KO protein or todisruption the formation of an alpha helices and beta sheets.

Protein of Interest

The term “protein of interest” (POI) as used herein refers to a proteinthat is produced by means of recombinant technology in a host cell. Morespecifically, the protein may either be a polypeptide not naturallyoccurring in the host cell, i.e. a heterologous protein, or else may benative to the host cell, i.e. a homologous protein to the host cell, butis produced, for example, by transformation with a self-replicatingvector containing the nucleic acid sequence encoding the POI, or uponintegration by recombinant techniques of one or more copies of thenucleic acid sequence encoding the POI into the genome of the host cell,or by recombinant modification of one or more regulatory sequencescontrolling the expression of the gene encoding the POI, e.g. of thepromoter sequence. In general, the proteins of interest referred toherein may be produced by methods of recombinant expression well knownto a person skilled in the art.

There is no limitation with respect to the protein of interest (POI).The POI is usually a eukaryotic or prokaryotic polypeptide, variant orderivative thereof. The POI can be any eukaryotic or prokaryoticprotein. The protein can be a naturally secreted protein or anintracellular protein, i.e. a protein which is not naturally secreted.The present invention also includes biologically active fragments ofproteins. In another embodiment, a POI may be an amino acid chain orpresent in a complex, such as a dimer, trimer, hetero-dimer, multimer oroligomer.

The protein of interest may be a protein used as nutritional, dietary,digestive, supplements, such as in food products, feed products, orcosmetic products. The food products may be, for example, bouillon,desserts, cereal bars, confectionery, sports drinks, dietary products orother nutrition products. Preferably, the protein of interest is a foodadditive.

In another embodiment, the protein of interest may be used in animalfeeds. The POI may be a detoxifying enzyme such as a mycotoxin degradingenzyme. A detoxifying enzyme means an enzyme which breaks down a toxinsuch to reduce its toxicity. Mycotoxins are toxic secondary metabolitesproduced by fungi that readily colonize crops and are oftencharacterized by the ability to harm crops and cause health problems forpeople and animals. Mycotoxin degrading enzymes include aflatoxindetoxizyme, zearalenone esterases, zearalenone lactonases, zearalenonehydrolase, fumonisin carboxylesterases, fumonisin aminotransferases,aminopolyol amine oxidases, deoxynivalenol expoxide hydrolases. The POImay also be an enzyme which degrades ochratoxin derivatives or ergotalkaloid. Ochratoxins are a group of mycotoxins produced as secondarymetabolites by several fungi of the Aspergillus or Penicillium familiesand are weak organic acids consisting of a derivative of an isocoumarin.There are three generally recognized ochratoxins, designated A, B and C.Ochratoxin A is the most abundant member of the ochratoxin family andhence the most commonly detected, but is also the most toxic. OchratoxinA (ochratoxin A) is a nephrotoxic, teratogenic, hepatotoxic, andcarcinogenic mycotoxin present in cereals and other starch rich foods.Ergot alkaloids are compounds containing amide bonds and include, forexample, ergocornine, ergocorninine, ergocristine, ergocristinine,ergocryptine, ergocryptinine, ergometrine, ergosine, ergotamine andergotaminine. These compounds are toxic to living organisms includinghumans and farm animals. Examples of such enzymes include ochratoxinamidase, ergotamine hydrolase, ergotamine amylase. Mycotoxin degradingenzymes in animal feed is useful in controlling mycotoxin contaminationof feed.

Further examples of POI include anti-microbial proteins, such aslactoferrin, lysozyme, lactoferricin, lactohedrin, kappa-casein,haptocorrin, lactoperoxidase, a milk protein, acute-phase proteins,e.g., proteins that are produced normally in production animals inresponse to infection, and small anti-microbial proteins such aslysozyme and lactoferrin. Other examples include bactericidal protein,antiviral proteins, acute phase proteins (induced in production animalsin response to infection), probiotic proteins, bacteriostatic protein,and cationic antimicrobial proteins.

“Feed” means any natural or artificial diet, meal or the like orcomponents of such meals intended or suitable for being eaten, taken in,digested, by a non-human animal. A “feed additive” generally refers tosubstances added in a feed. It typically include one or more compoundssuch as vitamins, minerals, enzymes and suitable carriers and/orexcipient. For the present invention, a food additive may be an enzymeor other proteins. Examples of enzymes which can be used as feedadditive include phytase, xylanase and β-glucanase. A “food” means anynatural or artificial diet meal or the like or components of such mealsintended or suitable for being eaten, taken in, digested, by a humanbeing.

A “food additive” is generally refers to substances added in a food. Ittypically includes one or more compounds such as vitamins, minerals,enzymes and suitable carriers and/or excipient. For the presentinvention, a food additive may be an enzyme or other proteins. Examplesof enzymes which can be used as food additive include protease, lipase,lactase, pectin methyl esterase, pectinase, transglutaminase, amylase,β-glucanase, acetolactate decarboxylase and laccase.

In some embodiments, the food additive is an anti-microbial protein,which includes, for example, (i) anti-microbial milk proteins (eitherhuman or non-human) lactoferrin, lysozyme, lactoferricin, lactohedrin,kappa-casein, haptocorrin, lactoperoxidase, alpha-1-antitrypsin, andimmunoglobulins, e.g., IgA, (ii) acute-phase proteins, such asC-reactive protein (CRP); lactoferrin; lysozyme; serum amyloid A (SAA);ferritin; haptoglobin (Hp); complements 2-9, in particular complement-3;seromucoid; ceruloplasmin (Cp); 15-keto-13,14-dihydro-prostaglandin F2alpha (PGFM); fibrinogen (Fb); alpha(1)-acid glycoprotein (AGP);alpha(1)-antitrypsin; mannose binding protein; lipoplysaccharide bindingprotein; alpha-2 macroglobulin and various defensins, (iii)antimicrobial peptides, such as cecropin, magainin, defensins,tachyplesin, parasin I.buforin I, PMAP-23, moronecidin, anoplin,gambicin, and SAMP-29, and (iv) other anti-microbial protein(s),including CAP37, granulysin, secretory leukocyte protease inhibitor,CAP18, ubiquicidin, bovine antimicrobial protein-1, Ace-AMP1,tachyplesin, big defensin, Ac-AMP2, Ah-AMP1, and CAP18.

A POI may be an enzyme. Preferred enzymes are those which can be usedfor industrial application, such as in the manufacturing of a detergent,starch, fuel, textile, pulp and paper, oil, personal care products, orsuch as for baking, organic synthesis, and the like. Examples of suchenzymes include protease, amylase, lipase, mannanase and cellulose forstain removal and cleaning; pullulanase amylase and amyloglucosidase forstarch liquefaction and saccharification; glucose isomerase for glucoseto fructose conversion; cyclodextrin-glycosyltransferase forcyclodextrin production; xylanase for xiscosity reduction in fuel andstarch; amylase, xylanase, lipase, phospholipase, glucose, oxidase,lipoxygenase, transglutaminase for dough stability and conditioning inbaking; cellulase in textile manufacturing for denim finishing andcotton softening; amylase for de-sizing of texile; pectate lyase forscouring; catalase for bleach termination; laccase for bleaching;peroxidase for excess dye removal; lipase, protease, amylase, xylanase,cellulose, in pulp and paper production; lipase for transesterificationand phospholipase for de-gumming in fat processing fats and oils; lipasefor resolution of chiral alcohols and amides in organic synthesis;acylase for synthesis of semisynthetic penicillin, nitrilase for thesynthesis of enantiopure carboxylic acids; protease and lipase forleather production; amyloglucosidase, glucose oxidase, and peroxidasefor the making personal care products (see Kirk et al., Current Opinionin Biotechnology (2002) 13:345-351)

Therapeutic Protein

A POI may be a therapeutic protein. A POI may be but is not limited to aprotein suitable as a biopharmaceutical substance like an antibody orantibody fragment, growth factor, hormone, enzyme, vaccine, or.

The POI may be a naturally secreted protein or an intracellular protein,i.e. a protein which is not naturally secreted. The present inventionalso provides for the recombinant production of functional homologues,functional equivalent variants, derivatives and biologically activefragments of naturally secreted or not naturally secreted proteins.Functional homologues are preferably identical with or correspond to andhave the functional characteristics of a sequence.

The POI may be structurally similar to the native protein and may bederived from the native protein by addition of one or more amino acidsto either or both the C- and N-terminal end or the side-chain of thenative protein, substitution of one or more amino acids at one or anumber of different sites in the native amino acid sequence, deletion ofone or more amino acids at either or both ends of the native protein orat one or several sites in the amino acid sequence, or insertion of oneor more amino acids at one or more sites in the native amino acidsequence. Such modifications are well known for several of the proteinsmentioned above.

Preferably, the protein of interest is a mammalian polypeptide or evenmore preferably a human polypeptide. Especially preferred therapeuticproteins, which refer to any polypeptide, protein, protein variant,fusion protein and/or fragment thereof which may be administered to amammal. It is envisioned but not required that therapeutic proteinaccording to the present invention is heterologous to the cell. Examplesof proteins that can be produced by the cell of the present inventionare, without limitation, enzymes, regulatory proteins, receptors,peptide hormones, growth factors, cytokines, scaffold binding proteins(e.g. anticalins), structural proteins, lymphokines, adhesion molecules,receptors, membrane or transport proteins, and any other polypeptidesthat can serve as agonists or antagonists and/or have therapeutic ordiagnostic use. Moreover, the proteins of interest may be antigens asused for vaccination, vaccines, antigen-binding proteins, immunestimulatory proteins. It may also be an antigen-binding fragment of anantibody, which can include any suitable antigen-binding antibodyfragment known in the art. For example, an antibody fragment may includebut not limited to Fv (a molecule comprising the VL and VH),single-chain Fv (scFV) (a molecule comprising the VL and VH connectedwith by peptide linker), Fab, Fab′, F(ab′)₂, single domain antibody(sdAb) (molecules comprising a single variable domain and 3 CDR), andmultivalent presentations thereof. The antibody or fragments thereof maybe murine, human, humanized or chimeric antibody or fragments thereof.Examples of therapeutic proteins include an antibody, polyclonalantibody, monoclonal antibody, recombinant antibody, antibody fragments,such as Fab′, F(ab′)₂, Fv, scFv, di-scFvs, bi-scFvs, tandem scFvs,bispecific tandem scFvs, sdAb, nanobodies, V_(H), and V_(L), or humanantibody, humanized antibody, chimeric antibody, IgA antibody, IgDantibody, IgE antibody, IgG antibody, IgM antibody, intrabody, minibodyor monobody.

Such therapeutic proteins include, but are not limited to, insulin,insulin-like growth factor, hGH, tPA, cytokines, e.g. interleukines suchas IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) alpha,IFN beta, IFN gamma, IFN omega or IFN tau, tumor necrosisfactor (TNF)TNF alpha and TNF beta, TRAIL; G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF.

In a preferred embodiment, the protein is an antibody. The term“antibody” is intended to include any polypeptide chain-containingmolecular structure with a specific shape that fits to and recognizes anepitope, where one or more non-covalent binding interactions stabilizethe complex between the molecular structure and the epitope. Thearchetypal antibody molecule is the immunoglobulin, and all types ofimmunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g.human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken,other avians, etc., are considered to be “antibodies.” Numerous antibodycoding sequences have been described; and others may be raised bymethods well-known in the art.

For example, antibodies or antigen binding fragments may be produced bymethods known in the art. Generally, antibody-producing cells aresensitized to the desired antigen or immunogen. The messenger RNAisolated from antibody producing cells is used as a template to makecDNA using PCR amplification. A library of vectors, each containing oneheavy chain gene and one light chain gene retaining the initial antigenspecificity, is produced by insertion of appropriate sections of theamplified immunoglobulin cDNA into the expression vectors. Acombinatorial library is constructed by combining the heavy chain genelibrary with the light chain gene library. This results in a library ofclones which co-express a heavy and light chain (resembling the Fabfragment or antigen binding fragment of an antibody molecule). Thevectors that carry these genes are co-transfected into a host cell. Whenantibody gene synthesis is induced in the transfected host, the heavyand light chain proteins self-assemble to produce active antibodies thatcan be detected by screening with the antigen or immunogen.

Antibody coding sequences of interest include those encoded by nativesequences, as well as nucleic acids that, by virtue of the degeneracy ofthe genetic code, are not identical in sequence to the disclosed nucleicacids, and variants thereof. Variant polypeptides can include amino acid(aa) substitutions, additions or deletions. The amino acid substitutionscan be conservative amino acid substitutions or substitutions toeliminate non-essential amino acids, such as to alter a glycosylationsite, or to minimize misfolding by substitution or deletion of one ormore cysteine residues that are not necessary for function. Variants canbe designed so as to retain or have enhanced biological activity of aparticular region of the protein (e.g., a functional domain, catalyticamino acid residues, etc). Variants also include fragments of thepolypeptides disclosed herein, particularly biologically activefragments and/or fragments corresponding to functional domains.Techniques for in vitro mutagenesis of cloned genes are known. Alsoincluded in the subject invention are polypeptides that have beenmodified using ordinary molecular biological techniques so as to improvetheir resistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable as a therapeutic agent.

Chimeric antibodies may be made by recombinant means by combining thevariable light and heavy chain regions (VK and VH), obtained fromantibody producing cells of one species with the constant light andheavy chain regions from another. Typically, chimeric antibodies utilizerodent or rabbit variable regions and human constant regions, in orderto produce an antibody with predominantly human domains. The productionof such chimeric antibodies is well known in the art, and may beachieved by standard means (as described, e.g., in U.S. Pat. No.5,624,659.

Humanized antibodies are engineered to contain even more human-likeimmunoglobulin domains, and incorporate only thecomplementarity-determining regions of the animal-derived antibody. Thisis accomplished by carefully examining the sequence of thehyper-variable loops of the variable regions of the monoclonal antibody,and fitting them to the structure of the human antibody chains. Althoughfacially complex, the process is straightforward in practice. See, e.g.,U.S. Pat. No. 6,187,287.

In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)2, or other fragments) may be synthesized.“Fragment” or minimal immunoglobulins may be designed utilizingrecombinant immunoglobulin techniques. For instance “Fv” immunoglobulinsfor use in the present invention may be produced by synthesizing avariable light chain region and a variable heavy chain region.Combinations of antibodies are also of interest, e.g. diabodies, whichcomprise two distinct Fv specificities.

Immunoglobulins may be modified post-translationally, e.g. to addchemical linkers, detectable moieties, such as fluorescent dyes,enzymes, substrates, chemiluminescent moieties and the like, or specificbinding moieties, such as streptavidin, avidin, or biotin, and the likemay be utilized in the methods and compositions of the presentinvention.

Further examples of therapeutic proteins include blood coagulationfactors (VII, VIII, IX), alkaline protease from Fusarium, calcitonin,CD4 receptor darbepoetin, DNase (cystic fibrosis), erythropoetin,eutropin (human growth hormone derivative), follicle stimulating hormone(follitropin), gelatin, glucagon, glucocerebrosidase (Gaucher disease),glucosamylase from A. niger, glucose oxidase from A. niger,gonadotropin, growth factors (GCSF, GMCSF), growth hormones(somatotropines), hepatitis B vaccine, hirudin, human antibody fragment,human apolipoprotein AI, human calcitonin precursor, human collagenaseIV, human epidermal growth factor, human insulin-like growth factor,human interleukin 6, human laminin, human proapolipoprotein AI, humanserum albumininsulin, insulin and muteins, insulin, interferon alpha andmuteins, interferon beta, interferon gamma (mutein), interleukin 2,luteinization hormone, monoclonal antibody 5T4, mouse collagen, OP-1(osteogenic, neuroprotective factor), oprelvekin (interleu kin11-agonist), organophosphohydrolase, PDGF-agonist, phytase, plateletderived growth factor (PDGF), recombinant plasminogen-activator G,staphylokinase, stem cell factor, tetanus toxin fragment C, tissueplasminogen-activator, and tumor necrosis factor (see Schmidt, ApplMicrobiol Biotechnol (2004) 65:363-372).

Leader Sequence

The protein of interest may be linked with a leader sequence whichcauses secretion of the POI from the host cell. The presence of such asecretion leader sequence in the expression vector is required when thePOI intended for recombinant expression and secretion is a protein whichis not naturally secreted and therefore lacks a natural secretion leadersequence, or its nucleotide sequence has been cloned without its naturalsecretion leader sequence. In general, any secretion leader sequenceeffective to cause secretion of the POI from the host cell may be usedin the present invention. The secretion leader sequence may originatefrom yeast source, e.g. from yeast α-factor such as MFa of Saccharomycescerevisiae, or yeast phosphatase, from mammalian or plant source, orothers. The selection of the appropriate secretion leader sequence isapparent to a skilled person. Alternatively, the secretion leadersequence can be fused to the nucleotide sequence encoding a POI intendedfor recombinant expression by conventional cloning techniques known to askilled person prior to cloning of the nucleotide sequence in theexpression vector or the nucleotide sequence encoding a POI comprising anatural secretion leader sequence is cloned in the expression vector. Inthese cases the presence of a secretion leader sequence in theexpression vector is not required.

Promoter

The sequence encoding POI may be operably linked to a promoter. The term“promoter” as used herein refers to a region that facilitates thetranscription of a particular gene. A promoter typically increases theamount of recombinant product expressed from a nucleotide sequence ascompared to the amount of the expressed recombinant product when nopromoter exists. A promoter from one organism can be utilized to enhancerecombinant product expression from a sequence that originates fromanother organism. The promoter can be integrated into a host cellchromosome by homologous recombination using methods known in the art(e.g. Datsenko et al, Proc. Natl. Acad. Sci. U.S.A., 97(12): 6640-6645(2000)). In addition, one promoter element can increase the amount ofproducts expressed for multiple sequences attached in tandem. Hence, onepromoter element can enhance the expression of one or more recombinantproducts.

The promoter could be an “inducible promoter” or “constitutivepromoter.” “Inducible promoter” refers to a promoter which can beinduced by the presence or absence of certain factors, and “constitutivepromoter” refers to an unregulated promoter that allows for continuoustranscription of its associated gene or genes.

In a preferred embodiment, the nucleotide sequences encoding the POI isdriven by an inducible promoter.

Many inducible promoters are known in the art. Many are described in areview by Gatz, Curr. Op. Biotech., 7: 168 (1996) (see also Gatz, Ann.Rev. Plant. Physiol. Plant Mol. Biol., 48:89 (1997)). Examples includetetracycline repressor system, Lac repressor system, copper-induciblesystems, salicylate-inducible systems (such as the PR1 a system),glucocorticoid-inducible (Aoyama et al., 1997), alcohol-induciblesystems, e.g., AOX promoters, and ecdysome-inducible systems. Alsoincluded are the benzene sulphonamide-inducible (U.S. Pat. No.5,364,780) and alcohol-inducible (WO 97/06269 and WO 97/06268) induciblesystems and glutathione S-transferase promoters.

Suitable promoter sequences for use with yeast host cells are describedin Mattanovich et al., Methods Mol. Biol. (2012) 824:329-58 and includeglycolytic enzymes like triosephosphate isomerase (TPI),phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase(GAPDH or GAP) and variants thereof, lactase (LAC) and galactosidase(GAL), P. pastoris glucose-6-phosphate isomerase promoter (PPGI), the3-phosphoglycerate kinase promoter (PPGK), the glycerol aldehydephosphate dehydrogenase promoter (PGAP), translation elongation factorpromoter (PTEF), and the promoters of P. pastoris enolase 1 (PENO1),triose phosphate isomerase (PTPI), ribosomal subunit proteins (PRPS2,PRPS7, PRPS31, PRPL1), alcohol oxidase promoter (PAOX) or variantsthereof with modified characteristics, the formaldehyde dehydrogenasepromoter (PFLD), isocitrate lyase promoter (PICL), alpha-ketoisocaproatedecarboxylase promoter (PTHI), the promoters of heat shock proteinfamily members (PSSA1, PHSP90, PKAR2), 6-Phosphogluconate dehydrogenase(PGND1), phosphoglycerate mutase (PGPM1), transketolase (PTKL1),phosphatidylinositol synthase (PPIS1), ferro-O2-oxidoreductase (PFET3),high affinity iron permease (PFTR1), repressible alkaline phosphatase(PPHO8), N-myristoyl transferase (PNMT1), pheromone responsetranscription factor (PMCM1), ubiquitin (PUBI4), single-stranded DNAendonuclease (PRAD2), the promoter of the major ADP/ATP carrier of themitochondrial inner membrane (PPET9) (WO2008/128701) and the formatedehydrogenase (FMD) promoter. The GAP promoter, AOX promoter or apromoter derived from GAP or AOX promoter is particularly preferred. AOXpromoters can be induced by methanol and are repressed by glucose.

Further examples of suitable promoters include Saccharomyces cerevisiaeenolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1),Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase (PGK), and the maltase gene promoter (MAL).

Other useful promoters for yeast host cells are described by Romanos etal, 1992, Yeast 8:423-488.

Suitable promoter sequences for use with E. coli include T7 promoter, T5promoter, tryptophan (trp) promoter, lactose (lac) promoter,tryptophan/lactose (tac) promoter, lipoprotein (lpp) promoter, and Aphage PL promoter in plasmids.

As shown in the examples, the inventors have discovered that when thegenes are underexpressed, the yield of the POI (SDZ-Fab or HyHEL-Fab) byat least 20% compared to the host cell without the underexpression.

In a preferred embodiment, underexpression of KO1 gene increases theyield of the model protein SDZ-Fab or HyHEL-Fab in the host cell by atleast 1%, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or atleast 200% or more, compared to the host cell prior to engineering.In a preferred embodiment, underexpression of KO2 gene increases theyield of the model protein SDZ-Fab or HyHEL-Fab in the host cell by atleast 1%, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or atleast 200% or more, compared to the host cell prior to engineering.In a preferred embodiment, underexpression of KO3 gene increases theyield of the model protein SDZ-Fab or HyHEL-Fab in the host cell by atleast 1%, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or atleast 200% or more, compared to the host cell prior to engineering.

It is envisioned that in preferred embodiments, when the host cell arecultured to produce the protein of interest, the cell underexpresses atleast one the polynucleotide encoding a KO protein having an amino acidsequence as shown in SEQ ID NO: 10, 11, 12 or a functional homologuethereof, wherein the functional homologue has at least 30% sequenceidentity to an amino acid sequence as shown in SEQ ID NO: 10, 11 or 12and expresses the protein of interest.

In a further aspect, the present invention provides the use of theengineered host cell for manufacturing a protein of interest. The hostcells can be advantageously used for introducing polynucleotidesencoding one or more POI(s), and thereafter can be cultured undersuitable conditions to express the POI. Details of such use aredescribed in the later section concerning methods of the presentinvention.

The POI encoding polynucleotide may be recombined in to the host cell byligating the relevant genes into a vector, and transforming the hostcell with the vectors. More teaching can be found in the followingsections of the application.

Generally, proteins of interest can be produced by culturing the hostcell in an appropriate medium, isolating the expressed POI from theculture, and purifying it by a method appropriate for the expressedproduct, in particular to separate the POI from the cell.

In another aspect, the present invention provides a method of increasingthe yield of a protein of interest in a host cell, comprisingunderexpressing at least one polynucleotide encoding a KO protein havingan amino acid sequence as shown in SEQ ID NO: 10, 11, 12 or a functionalhomologue thereof, wherein the functional homologue has at least 30%sequence identity to an amino acid sequence as shown in SEQ ID NO: 10,11 or 12.

As used herein, the term “increasing the yield of a protein of interestin a host cell” means that the yield of the protein of interest isincreased when compared to the same cell expressing the same POI underthe same culturing conditions, however, without the underexpression ofthe knockout protein(s).

As will be appreciated by a skilled person in the art, theunderexpression of the present KO protein(s) have been shown to increaseproduct yield of POI. Therefore, for a given host cell which expressed aPOI with a level that should be increased, it is possible to apply thepresent invention by underexpressing the present KO protein(s).

In yet a further aspect, the present invention provides a method ofincreasing the yield of a protein of interest in a host cell. The methodcomprises (i) engineering the host cell to underexpress at least one,polynucleotide encoding a KO protein having an amino acid sequence asshown in SEQ ID NO: 10, 11, 12 or a functional homologue thereof,wherein the functional homologue has at least 30% sequence identity toan amino acid sequence as shown in SEQ ID NO: 10, 11 or 12, (ii)recombining in said host cell a heterologous polynucleotide encoding aprotein of interest, and (iii) culturing said host cell under suitableconditions to express the protein of interest.

As used herein, the term “recombining” as used herein means that a hostcell of the present invention is equipped with a heterologouspolynucleotide encoding a protein of interest, i.e., a host cell of thepresent invention is engineered to contain a heterologous polynucleotideencoding a protein of interest. This can be achieved, e.g., bytransformation or transfection or any other suitable technique known inthe art for the introduction of a polynucleotide into a host cell.

Procedures used to manipulate polynucleotide sequences, e.g. coding forthe POI, the promoter, enhancers, leaders, etc., are well known topersons skilled in the art, e.g. described by J. Sambrook et al.,Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, New York (2001).

A foreign or target polynucleotide such as the polynucleotides encodingthe POI or helper protein can be inserted into the chromosome by variousmeans, e.g., by homologous recombination or by using a hybridrecombinase that specifically targets sequences at the integrationsites. The foreign or target polynucleotide described above is typicallypresent in a vector (“inserting vector”). These vectors are typicallycircular and linearized before used for homologous recombination. As analternative, the foreign or target polynucleotides may be DNA fragmentsjoined by fusion PCR or synthetically constructed DNA fragments whichare then recombined into the host cell. In addition to the homologyarms, the vectors may also contain markers suitable for selection orscreening, an origin of replication, and other elements

Polynucleotides encoding the target sequence may also be present on anexpression vector. Such vectors are known in the art and alreadydescribed above. In expression vectors, a promoter is placed upstream ofthe gene encoding the heterologous protein and regulates the expressionof the gene. Multi-cloning vectors are especially useful due to theirmulti-cloning site. For expression, a promoter is generally placedupstream of the multi-cloning site. A vector for integration of thepolynucleotides encoding the POI may be constructed either by firstpreparing a DNA construct containing the entire DNA sequence coding forthe POI and subsequently inserting this construct into a suitableexpression vector, or by sequentially inserting DNA fragments containinggenetic information for the individual elements, such as the leadersequence, the target DNA sequence, followed by ligation. As analternative to restriction and ligation of fragments, recombinationmethods based on attachment (att) sites and recombination enzymes may beused to insert DNA sequences into a vector. Such methods are described,for example, by Landy (1989) Ann. Rev. Biochem. 58:913-949; and areknown to those of skill in the art.

Host cells according to the present invention can be obtained byintroducing a vector or plasmid comprising the target polynucleotidesequences into the cells. Techniques for transfecting or transformingeukaryotic cells or transforming prokaryotic cells are well known in theart. These can include lipid vesicle mediated uptake, heat shockmediated uptake, calcium phosphate mediated transfection (calciumphosphate/DNA co-precipitation), viral infection, particularly usingmodified viruses such as, for example, modified adenoviruses,microinjection and electroporation. For prokaryotic transformation,techniques can include heat shock mediated uptake, bacterial protoplastfusion with intact cells, microinjection and electroporation. Techniquesfor plant transformation include Agrobacterium mediated transfer, suchas by A. tumefaciens, rapidly propelled tungsten or goldmicroprojectiles, electroporation, microinjection and polyethylyneglycol mediated uptake. The DNA can be single or double stranded, linearor circular, relaxed or supercoiled DNA. For various techniques fortransfecting mammalian cells, see, for example, Keown et al. (1990)Processes in Enzymology 185:527-537.

In a further aspect, the present invention provides a method ofmanufacturing a protein of interest in a host cell comprising (i)providing a host cell engineered to underexpress at least onepolynucleotide encoding a protein having an amino acid sequence as shownin SEQ ID NO: 10, 11, 12 or a functional homologue thereof, wherein thefunctional homologue has at least 30% sequence identity to an amino acidsequence as shown in SEQ ID NO: 10, 11, 12, wherein said host cellcomprises a heterologous polynucleotide encoding a protein of interest;and (ii) culturing the host cell under suitable conditions to expressthe protein of interest. It is understood that the methods disclosedherein may further include cultivating said recombinant host cells underconditions permitting the expression of the POI. A recombinantlyproduced POI can then be isolated from the cell or the cell culturemedium, depending on the nature of the expression system and theexpressed protein, e.g. whether the protein is fused to a signal peptideand whether the protein is soluble or membrane-bound. As will beunderstood by the skilled artisan, cultivation conditions will varyaccording to factors that include the type of host cell, in particularthe expression vector employed. Signal peptides generally contain apositively charged N-terminus followed by a hydrophobic core, followedby a recognition site for an enzyme known as signal peptidase. Thisenzyme cleaves the signal peptide from the protein during translocation.The protein is transported from the endoplasmic reticulum to the Golgiapparatus, and then follows one of a number of routes in the secretorypathway, depending on the nature of the protein. The protein may besecreted into the culture medium or may be retained on the cell surface,for example. Certain receptors that comprise extracellular,transmembrane, and cytoplasmic domains are examples of proteins that maybe retained on the cell membrane, with only the extracellular domainlocated outside the cell. The leader sequences of certain secretedproteins comprise peptides that are located C-terminal to the signalpeptide and are processed from the mature protein of interest subsequentto cleavage of the signal peptide. Such leaders often are referred to asprepro peptides, wherein the pre region is the signal sequence and thepro region designates the remainder of the leader.

One example is the yeast α-factor leader, which contains a signalpeptide (including a C-terminal signal peptidase recognition siteAlaLeuAla) followed by a pro region containing a basic amino acid pairLysArg that constitutes a KEX2 protease processing site, immediatelyfollowed by a peptide GluAlaGluAla at the C-terminus of the pro region.Processing of this leader involves removal of the signal peptide bysignal peptidase, followed by cleavage between the Lys and Arg residuesby KEX2 protease. The GluAlaGluAla residues are subsequently removed bya peptidase that is the product of the STE13 gene (Julius et al., Cell(1983) 32:839). The yeast α-factor leader is described in U.S. Pat. No.4,546,082. Signal peptides derived from proteins naturally secreted byyeast cells have been employed in recombinant expression systems forproduction of heterologous proteins in yeast. The use of mammaliansignal peptides in yeast expression systems also has been reported,although certain of the mammalian signal peptides were not effective inpromoting secretion of heterologous proteins in yeast.

The phrase culturing under “suitable conditions such that a desiredpolypeptide is expressed” refers to maintaining and/or growingmicroorganisms under conditions (e.g., temperature, pressure, pH,duration, etc.) appropriate or sufficient to obtain the desired compoundor to obtain desired polypeptide.

In preferred embodiments the host cell also expresses a helper protein.Helper proteins are described in later sections of the application.Using different promoters and/or copies and/or integration sites for thehelper gene(s) and/or the POI(s), the expression of the helper genes canbe controlled with respect to time point and strength of induction inrelation to the expression of the POI(s). For example, prior toinduction of POI expression, the helper protein(s) may be firstexpressed. This has the advantage that the helper protein(s) are alreadypresent at the beginning of POI translation. Alternatively, the helperprotein(s) and POI(s) can be induced at the same time.

A host cell according to the invention obtained by transformation withthe POI genes may preferably first be cultivated at conditions to growefficiently to a large cell number without the burden of expressing aheterologous protein. When the cells are prepared for POI expression,suitable cultivation conditions are selected and optimized to producethe POI.

An inducible promoter may be used that becomes activated as soon as aninductive stimulus is applied, to direct transcription of the gene underits control. Under growth conditions with an inductive stimulus, thecells usually grow more slowly than under normal conditions, but sincethe culture has already grown to a high cell number in the previousstage, the culture system as a whole produces a large amount of theheterologous protein. An inductive stimulus is preferably the additionof an appropriate agents (e.g.: methanol for the AOX-promoter) or thedepletion of an appropriate nutrient (e.g., methionine for theMET3-promoter). Also, the addition of ethanol, methylamine, cadmium orcopper as well as heat or an osmotic pressure increasing agent caninduce the expression.

It is preferred to cultivate the hosts according to the invention in abioreactor under optimized growth conditions to obtain a cell density ofat least 1 g/L, preferably at least 10 g/L cell dry weight, morepreferably at least 50 g/L cell dry weight. It is advantageous toachieve such yields of POI not only on a laboratory scale, but also on apilot or industrial scale.

According to the present invention, due to underexpression of the KOprotein, the POI is obtainable in high yields, which can be measured inmg POI/g dry biomass, in the range of 1 to 200, such as 50-200, such as100-200 in the laboratory, pilot and industrial scale is feasible. Thespecific yield of a production host according to the inventionpreferably provides for an increase of at least 1.1 fold, morepreferably at least 1.2 fold, at least 1.3 or at least 1.4 fold, in somecases an increase of more than 2 fold can be shown, when compared to theexpression of the product without the underexpression of helperproteins.

The host cell according to the invention may be tested for itsexpression/secretion capacity or yield by standard tests, e.g. ELISA,activity assays, HPLC, Surface Plasmon Resonance (Biacore), WesternBlot, capillary electrophoresis (Caliper) or SDS-Page.

Preferably, the cells are cultivated in a mineral medium with a suitablecarbon source, thereby further simplifying the isolation processsignificantly. By way of example, the mineral medium contains anutilizable carbon source (e.g. glucose, glycerol or methanol), saltscontaining the macro elements (potassium, magnesium, calcium, ammonium,chloride, sulphate, phosphate) and trace elements (copper, iodide,manganese, molybdate, cobalt, zinc, and iron salts, and boric acid).

In the case of yeast cells, the cells may be transformed with one ormore of the above-described expression vector(s) or plasmid(s), mated toform diploid strains, and cultured in conventional nutrient mediamodified as appropriate for inducing promoters, selecting transformantsor amplifying the genes encoding the desired sequences. A number ofminimal media suitable for the growth of yeast are known in the art. Anyof these media may be supplemented as necessary with salts (such assodium chloride, calcium, magnesium, and phosphate), buffers (such asHEPES, citric acid and phosphate buffer), nucleosides (such as adenosineand thymidine), antibiotics, trace elements, vitamins, and glucose or anequivalent energy source. Any other necessary supplements may also beincluded at appropriate concentrations that would be known to thoseskilled in the art. The culture conditions, such as temperature, pH andthe like, are those previously used with the host cell selected forexpression and are known to the ordinarily skilled artisan. Cell cultureconditions for other type of host cells are also known and can bereadily determined by the artisan. Descriptions of culture media forvarious microorganisms are for example contained in the handbook “Manualof Methods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981).

Cells can be cultured (e.g., maintained and/or grown) in liquid mediaand preferably are cultured, either continuously or intermittently, byconventional culturing methods such as standing culture, test tubeculture, shaking culture (e.g., rotary shaking culture, shake flaskculture, etc.), aeration spinner culture, or fermentation. In someembodiments, cells are cultured in shake flasks or deep well plates. Inyet other embodiments, cells are cultured in a bioreactor (e.g., in abioreactor cultivation process). Cultivation processes include, but arenot limited to, batch, fed-batch and continuous methods of cultivation.The terms “batch process” and “batch cultivation” refer to a closedsystem in which the composition of media, nutrients, supplementaladditives and the like is set at the beginning of the cultivation andnot subject to alteration during the cultivation; however, attempts maybe made to control such factors as pH and oxygen concentration toprevent excess media acidification and/or cell death. The terms“fed-batch process” and “fed-batch cultivation” refer to a batchcultivation with the exception that one or more substrates orsupplements are added (e.g., added in increments or continuously) as thecultivation progresses. The terms “continuous process” and “continuouscultivation” refer to a system in which a defined cultivation media isadded continuously to a bioreactor and an equal amount of used or“conditioned” media is simultaneously removed, for example, for recoveryof the desired product. A variety of such processes has been developedand is well-known in the art.

In some embodiments, cells are cultured for about 12 to 24 hours, inother embodiments, cells are cultured for about 24 to 36 hours, about 36to 48 hours, about 48 to 72 hours, about 72 to 96 hours, about 96 to 120hours, about 120 to 144 hours, or for a duration greater than 144 hours.In yet other embodiments, culturing is continued for a time sufficientto reach desirable production yields of POI.

The above mentioned methods may further comprise a step of isolating theexpressed POI. If the POI is secreted from the cells, it can be isolatedand purified from the culture medium using state of the art techniques.Secretion of the POI from the cells is generally preferred, since theproducts are recovered from the culture supernatant rather than from thecomplex mixture of proteins that results when cells are disrupted torelease intracellular proteins. A protease inhibitor, such as phenylmethyl sulfonyl fluoride (PMSF) may be useful to inhibit proteolyticdegradation during purification, and antibiotics may be included toprevent the growth of adventitious contaminants. The composition may beconcentrated, filtered, dialyzed, etc., using methods known in the art.Alternatively, cultured host cells may also be ruptured sonically ormechanically, enzymatically or chemically to obtain a cell extractcontaining the desired POI, from which the POI may be isolated andpurified.

As isolation and purification methods for obtaining the POI may be basedon methods utilizing difference in solubility, such as salting out andsolvent precipitation, methods utilizing difference in molecular weight,such as ultrafiltration and gel electrophoresis, methods utilizingdifference in electric charge, such as ion-exchange chromatography,methods utilizing specific affinity, such as affinity chromatography,methods utilizing difference in hydrophobicity, such as reverse phasehigh performance liquid chromatography, and methods utilizing differencein isoelectric point, such as isoelectric focusing may be used. Specificpurification steps are preferably employed to remove any helper proteinthat is also expressed and would contaminate the POI preparation.

The isolated and purified POI can be identified by conventional methodssuch as Western Blotting or specific assays for its activity. Thestructure of the purified POI can be defined by amino acid analysis,amino-terminal analysis, primary structure analysis, and the like. It ispreferred that the POI is obtainable in large amounts and in a highpurity level, thus meeting the necessary requirements for being used asan active ingredient in pharmaceutical compositions or as feed or foodadditive.

Helper Proteins

The host cell of the present invention may additionally comprise atleast one polynucleotide encoding a helper protein. Any helper proteinknown in the art can be used.

A “helper proteins” as used in the present invention means a proteinwhich enhances the expression and/or secretion of a protein of interest.This term should be understand broadly and should not be limited tochaperons or chaperon-like proteins.

A helper protein may for example be a chaperone. A chaperone generallyhas the function of binding to and stabilising an otherwise unstableconformer of another protein. By controlled binding and release, achaperone facilitates its correct fate in vivo, be it folding,oligomeric assembly, transport to a particular subcellular compartment,or disposal by degradation. Chaperone proteins of this type are known inthe art, for example in the Stanford Genome Database (SGD),http://db.yeastgenome.org or http://www.yeastgenome.org. Preferredchaperones are eukaryotic chaperones, especially preferred chaperonesare yeast chaperones, including AHA1, CCT2, CCT3, CCT4, CCT5, CCT6,CCT1, CCT8, CNS1, CPR3, CPR6, ERO1, EUG1, FMO1, HSP10, HSP12, HSP104,HSP26, HSP30, HSP42, HSP60, HSP78, HSP82, JEM1, MDJ1, MDJ2, MPD1, MPD2,PDI1, PFD1, ABC1, APJ1, ATP11, ATP12, BTT1, CDC37, CPR7, HSC82, KAR2,LHS1, MGE1, MRS11, NOB1, ECM10, SSA1, SSA2, SSA3, SSA4, SSC1, SSE2,SIL1, SLS1, ORM1, ORM2, PERI, PTC2, PSE1, UBI4 and HAC1 or a truncatedintronless HAC1 (Valkonen et al. 2003, Applied Environ Micro., 69,2065), as well as TIM9, PAM18 (also known as TIM14) and TCP1 (also knownas CCT1). For example, the host cell may comprise a polynucleotideencoding BMH2, BFR2, COG6, COY1, CUP5, IMH1, KIN2, SEC31, SSA4, SSE1 orKAR2 (WO2008128701).

Preferably, the host cell comprises at least one polynucleotide encodinga helper protein of the present invention, namely, HP1, HP2, HP3, HP4,HP5, HP6, HP7, HP8, HP9, HP10 or functional homologues thereof. A helperprotein of the present invention comprises the amino acid sequences ofSEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 162 and functional homologuesthereof. A helper protein of the present invention can be encoded by thenucleotide sequences of SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21,or 163. Thus, the term “helper protein” when used in the context ofHP1-HP10 is meant to encompass HP1, HP2, HP3, HP4, HP5, HP6, HP7, HP8,HP9 and HP 10 as well as functional homologues of HP1, HP2, HP3, HP4,HP5, HP6, HP7, HP8, HP9, and HP10, respectively.

As used herein, such helper protein has an amino acid sequence having atleast 30%, such as at least 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or 100%, sequence identity to an amino acid sequence asshown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 162. The amino acidsequences of the helper proteins HP1 to HP10 are listed in SEQ ID NO: 1,2, 3, 4, 5, 6, 7, 8, 9, or 162.

The helper proteins of the present invention were originally isolatedfrom Pichia pastoris CBS7435 strain. The methylotrophic yeast Pichiapastoris (Komagataella phaffii) CBS7435 is the parental strain ofcommonly used P. pastoris recombinant protein production hosts. Itscomplete genomic sequence is described in Küberl et al. (J Biotechnol.(2011) 154(4):312-20). These genes encoding the helper proteinsidentified herein have so far not been associated with a beneficialeffect on protein yield.

The helper proteins may be native to the species or the genus of thehost cell or taken or derived from other prokaryotic or eukaryoticorganisms. The foreign DNA sequences encoding the helper proteins may beobtained from a variety of sources, preferably from a plant, insect,fungal or mammalian species, preferably from the class ofSaccharomycetes, preferably from the order of Saccharomycetales,preferably from the family of Saccharomycetaceae, and preferably fromthe genus of Komagataella. Homologous nucleotide sequences from otherorganisms such as vertebrates may be used.

The amino acid sequences of each helper proteins and their correspondingdesignations in the Examples are listed in Table 2 below:

TABLE 2 Helper protein (HP) and alternative Amino acid designationDesignation (in sequences Polynucleotide Designation in the (‘xx’)analogy to S. cerevisiae) (SEQ ID NO:) (SEQ ID NO:) Examples (see Table7) HP1 (‘56’) SEQ ID NO: 4 SEQ ID NO: 16 PP7435_Chr3-0607 HP2 (‘2’) SEQID NO: 1 SEQ ID NO: 13 PP7435_Chr3-0933 HP3 (‘3’) SBH1 SEQ ID NO: 2 SEQID NO: 14 PP7435_Chr2-0220 HP4 (‘27’) CPR6 SEQ ID NO: 3 SEQ ID NO: 15PP7435_Chr3-0639 HP5 (‘4’) MXR2 SEQ ID NO: 5 SEQ ID NO: 17PP7435_Chr4-0108 HP6 (‘54’) MDR1 SEQ ID NO: 7 SEQ ID NO: 19PP7435_Chr1-1225 HP7 (‘55’) SEQ ID NO: 8 SEQ ID NO: 20 PP7435_Chr1-0667HP8 (‘40’) — SEQ ID NO: 6 SEQ ID NO: 18 PP7435_Chr1-1232 HP9 (‘60’) —SEQ ID NO: 9 SEQ ID NO: 21 PP7435_Chr4-0448 HP10 (‘34’) SEC61 SEQ ID NO:162 SEQ ID NO: 163 PP7435_Chr1-0204

In particular, out of a screen for secretion helper factors in Pichiapastoris 55 candidate genes were identified which were validated bywet-lab expression experiments in order to select prime candidate genes.A prime candidate gene is one which enhances yield of the two modelproteins SDZ-Fab#9 and/or HyHEL-Fab#8 as described herein to an extentof 20% or more in comparison to a P. pastoris strain not over-expressingthe prime candidate gene, i.e., a host cell prior to engineering asdescribed herein. To this end, out of the 55 genes, 9 prime candidateswere identified. 4 prime candidates, i.e. PP7435_Chr3-0607,PP7435_Chr3-0933, PP7435_Chr2-0220 (SBH1), PP7435_Chr3-0639 (CPR6)showed more than 20% secretion and/or expression enhancement of bothmodel proteins SDZ-Fab and HyHEL Fab and 5 further candidates(PP7435_Chr4-0108 (MXR2), PP7435_Chr1-1232, PP7435_Chr1-1225 (MDR1),PP7435_Chr1-0667, PP7435_Chr4-0448) showed >20-30% secretion yieldenhancement for one of the two model proteins SDZ-Fab and HyHEL Fab,respectively, when being overexpressed.

Basis for the screen was a direct comparison between the transcriptionalprofile of a P. pastoris producer strain and a P. pastoris non-producerstrain which resulted in the identification of 55 genes. These genesbelong to various metabolic pathways and have different functions or mayeven have an unknown function. Thus, no guidance as to the nature of thethus-obtained helper factors is apparent from the genes or proteinsencoded thereby. Accordingly, tests have to be performed in order tovalidate whether or not a potential secretion helper factor is indeed ahelper factor under real conditions. However, it is prima facie notapparent that a potential helper factor is indeed a helper factor,merely because it was identified by its enhanced transcript appearancein a producer strain. In fact, it may be that a potential helper factorhas no positive effect on protein secretion or may even have a negativeeffect. This was indeed observed by the inventors, since two genesencoding chaperones (SCJ1, HCH1) identified among the 55 genes, fromwhich it could be reasonably expected to enhance secretion, did theopposite. Thus, tests for each gene are required, but no guidance existsas to which protein encoded by one of the approx. 60 genes is indeed asecretion helper factor.

Hence, the finding of a “true” secretion helper factor is not astraightforward matter, but an inventive choice for which no guidance isavailable or apparent from the mere gene/protein sequence or thefunction of a protein identified in a screen as done by the presentinventors.

What is also unusual about the prime candidate genes that have to beoverexpressed as is described herein—they are not typical secretionhelper factors, i.e., genes which encode a protein having a functionthat is not deemed to play a role in protein secretion or even have anunknown function.

In a preferred embodiment, the host cell is further engineered toexpress or overexpress at least one, such as at least 2, 3, 4, 5, 6, 7,8, or at least 9 polynucleotide encoding a helper protein having anamino acid sequence as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or162 or a functional homologue thereof, wherein the functional homologuehas having at least 30% sequence identity to an amino acid sequence asshown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 162.

Preferably, the present invention provides a recombinant host cell formanufacturing a protein of interest, wherein the host cell is engineeredto overexpress a polynucleotide encoding a helper protein having anamino acid having at least 40% sequence identity to SEQ ID NO: 1, 45%sequence identity to SEQ ID NO: 2, 50% sequence identity to SEQ ID NO:3, 45% sequence identity to SEQ ID NO: 4, 50% sequence identity to SEQID NO: 5, 45% sequence identity to SEQ ID NO: 6, 40% sequence identityto SEQ ID NO: 7, 40% sequence identity to SEQ ID NO: 8, 40% sequenceidentity to SEQ ID NO: 9, or 45% sequence identity to SEQ ID NO: 162.Such a host cell is applied in the methods and uses described herein.

Preferably, the host cell is engineered to express or overexpress atleast one polynucleotide encoding a helper protein having an amino acidsequence having at least 30% sequence identity to an amino acid sequenceas shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 162. If thepolynucleotide(s) already exist in the host cell, the host cell canmanipulated in a way such that they are overexpressed, as will bedescribed later.

In a preferred embodiment, 2, 3, 4, 5, 6, 7, 8 9 or more types of helperproteins disclosed by present invention are overexpressed. For example,the host cell can be engineered to overexpress 2, 3, 4, 5, 6, 7, 8 ormore of helper proteins selected from SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8,9, or 162 or functional homologues thereof, where a functional homologuethereof has an amino acid having at least 30% sequence identity to SEQID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 162.

The term “overexpress” generally refers to any amount greater than orequal to an expression level exhibited by a reference standard. Theterms “overexpress,” “overexpressing,” “overexpressed” and“overexpression” in the present invention refer an expression of a geneproduct or a polypeptide at a level greater than the expression of thesame gene product or polypeptide prior to a genetic alteration of thehost cell or in a comparable host which has not been genetically alteredat defined conditions. If a host cell does not comprise a given geneproduct, it is possible to introduce the gene product into the host cellfor expression; in this case, any detectable expression is encompassedby the term “overexpression.” However, it is not required, althoughpreferred, that the host cell overexpresses the helper protein HP1-HP10or functional homologues thereof.

Overexpression of Helper Proteins by the Host Cell

Overexpression can be achieved in any ways known to a skilled person inthe art. In general, it can be achieved by increasingtranscription/translation of the gene, e.g. by increasing the copynumber of the gene or altering or modifying regulatory sequences orsites associated with expression of a gene. For example, the gene can beoperably linked to a strong constitutive promoter and/or strongubiquitous promoter in order to reach high expression levels. Suchpromoters can be endogenous promoters or recombinant promoters.Alternatively, it is possible to remove regulatory sequences such thatexpression becomes constitutive. One can substitute a promoter with aheterologous promoter which increases expression of the gene or leads toconstitutive expression of the gene. Using inducible promotersadditionally makes it possible to increase the expression in the courseof cultivation. Furthermore, overexpression can also be achieved by, forexample, modifying the chromosomal location of a particular gene,altering nucleic acid sequences adjacent to a particular gene such as aribosome binding site or transcription terminator, introducing aframe-shift in the open reading frame, modifying proteins (e.g.,regulatory proteins, suppressors, enhancers, transcriptional activatorsand the like) involved in transcription of the gene and/or translationof the gene product, or any other conventional means of deregulatingexpression of a particular gene routine in the art (including but notlimited to use of antisense nucleic acid molecules, for example, toblock expression of repressor proteins or deleting or mutating the genefor a transcriptional factor which normally represses expression of thegene desired to be overexpressed. Prolonging the life of the mRNA mayalso improve the level of expression. For example, certain terminatorregions may be used to extend the half-lives of mRNA (Yamanishi et al.,Biosci. Biotechnol. Biochem. (2011) 75:2234 and US 2013/0244243). Ifmultiple copies of genes are included, the genes can either be locatedin plasmids of variable copy number or integrated and amplified in thechromosome. If the host cell does not comprise the gene product encodingthe helper protein, it is possible to introduce the gene product intothe host cell for expression. In this case, “overexpression” meansexpressing the gene product using any methods known to a skilled personin the art.

The overexpression of the polypeptide encoding the helper proteins, canbe achieved by other methods known in the art, for example bygenetically modifying their endogenous regulatory regions, as describedby Marx et al., 2008 (Marx, H., Mattanovich, D. and Sauer, M. MicrobCell Fact 7 (2008): 23), and Pan et al., 2011 (Pan et al., FEMS YeastRes. (2011) May; (3):292-8.), such methods include, for example,integration of a recombinant promoter that increases expression of thehelper proteins. Transformation is described in Cregg et al. (1985) Mol.Cell. Biol. 5:3376-3385.

Those skilled in the art will find relevant instructions in Martin etal. (Bio/Technology 5, 137-146 (1987)), Guerrero et al. (Gene 138, 35-41(1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)),Eikmanns et al. (Gene 102, 93-98 (1991)), EP 0 472 869, U.S. Pat. No.4,601,893, Schwarzer and Puhler (Bio/Technology 9, 84-87 (1991)),Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132(1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)),WO 96/15246, Malumbres et al. (Gene 134, 15-24 (1993)), JP-A-10-229891,Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998))and Makrides (Microbiological Reviews 60, 512-538 (1996)), inter alia,and in well-known textbooks on genetics and molecular biology.

According to a preferred embodiment, the polynucleotide encoding thehelper protein can be presented in a single copy or in multiple copiesper cell. The copies may be adjacent to or distant from each other.According to another preferred embodiment, the method of the inventionemploys recombinant nucleotide sequences encoding the helper proteinsprovided on one or more plasmids suitable for integration into thegenome of the host cell, in a single copy or in multiple copies percell. The copies may be adjacent to or distant from each other.Overexpression can be achieved by expressing multiple copies of thepolynucleotide, such as 2, 3, 4, 5, 6 or more copies of saidpolynucleotide per host cell.

The recombinant nucleotide sequence encoding the POI(s), as well asthose encoding the helper proteins, may also be provided on one or moreautonomously replicating plasmids in a single copy or in multiple copiesper cell.

Alternatively, the recombinant nucleotide sequence encoding the POI andthe recombinant nucleotide sequence encoding a protein that increasesprotein secretion are present on the same plasmid in single copy ormultiple copies per cell.

The polynucleotide encoding the helper protein is preferably integratedinto the genome of the host cell. The term “genome” generally refers tothe whole hereditary information of an organism that is encoded in theDNA (or RNA for certain viral species). It may be present in thechromosome, on a plasmid or vector, or both. Preferably, thepolynucleotide encoding the helper protein is integrated into thechromosome of said cell.

The polynucleotide encoding the helper protein may be integrated in itsnatural locus. “Natural locus” means the location on a specificchromosome, where the polynucleotide encoding the helper protein islocated, for example at the natural locus of HP1 to 9 as shown inTable 1. However, in another embodiment, the polynucleotide encoding thehelper protein is present in the genome of the host cell not at theirnatural locus, but integrated ectopically. The term “ectopicintegration” means the insertion of a nucleic acid into the genome of amicroorganism at a site other than its usual chromosomal locus, i.e.,predetermined or random integration. In another embodiment, thepolynucleotide encoding the helper protein is integrated into thenatural locus and ectopically. Heterologous recombination can be used toachieve random or non-targeted integration. Heterologous recombinationrefers to recombination between DNA molecules with significantlydifferent sequences. Methods of recombinations are known in the art andfor example described in Boer et al., Appl Microbiol Biotechnol (2007)77:513-523. One may also refer to Principles of Gene Manipulation andGenomics by Primrose and Twyman (7th edition, Blackwell Publishing 2006)for genetic manipulation of yeast cells.

For yeast cells, the polynucleotide encoding the helper protein and/orthe polynucleotide encoding the POI may be inserted into a desiredlocus, such as AOX1, GAP, ENO1, TEF, HIS4 (Zamir et al., Proc. NatLAcad. Sci. USA (1981) 78(6):3496-3500), HO (Voth et al. Nucleic AcidsRes. 2001 Jun. 15; 29(12): e59), TYR1 (Mirisola et al., Yeast 2007; 24:761-766), His3, Leu2, Ura3 (Taxis et al., BioTechniques (2006)40:73-78), Lys2, ADE2, TRP1, GAL1, ADH1 or on the integration of 5Sribosomal RNA gene.

In other embodiments, the polynucleotide encoding the helper proteinand/or the polynucleotide encoding the POI can be integrated in aplasmid or vector. The terms “plasmid” and “vector” include autonomouslyreplicating nucleotide sequences as well as genome integratingnucleotide sequences. A skilled person is able to employ suitableplasmids or vectors depending on the host cell used.

Preferably, the plasmid is a eukaryotic expression vector, preferably ayeast expression vector.

Preferably, the plasmid is a eukaryotic expression vector, preferably ayeast expression vector. Examples of plasmids using yeast as a hostinclude YIp type vector, YEp type vector, YRp type vector, YCp typevector, pGPD-2, pAO815, pGAPZ, pGAPZα, pHIL-D2, pHIL-S1, pPIC3.5K,pPIC9K, pPICZ, pPICZα, pPIC3K, pHWO10, pPUZZLE and 2 μm plasmids. Suchvectors are known and are for example described in Cregg et al., MolBiotechnol. (2000) 16(1):23-52.

Plasmids can be used for the transcription of cloned recombinantnucleotide sequences, i.e. of recombinant genes and the translation oftheir mRNA in a suitable host organism. Plasmids can also be used tointegrate a target polynucleotide into the host cell genome by methodsknown in the art, such as described by J. Sambrook et al., MolecularCloning: A Laboratory Manual (3rd edition), Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, New York (2001). A“plasmid” usually comprises an origin for autonomous replication in thehost cells, selectable markers, a number of restriction enzyme cleavagesites, a suitable promoter sequence and a transcription terminator,which components are operably linked together. The polypeptide codingsequence of interest is operably linked to transcriptional andtranslational regulatory sequences that provide for expression of thepolypeptide in the host cells.

Most plasmids exist in only one copy per bacterial cell. Some plasmids,however, exist in higher copy numbers. For example, the plasmid ColE1typically exists in 10 to 20 plasmid copies per chromosome in E. coli.If the nucleotide sequences of the present invention are contained in aplasmid, the plasmid may have a copy number of 20-30, 30-100 or more perhost cell. With a high copy number of plasmids, it is possible tooverexpress helper proteins by the cell.

Large numbers of suitable plasmids or vectors are known to those ofskill in the art and many are commercially available. Examples ofsuitable vectors are provided in Sambrook et al, eds., MolecularCloning: A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring HarborLaboratory (1989), and Ausubel et al, eds., Current Protocols inMolecular Biology, John Wiley & Sons, Inc., New York (1997).

A vector or plasmid of the present invention encompass yeast artificialchromosome, which refers to a DNA construct that can be geneticallymodified to contain a heterologous DNA sequence (e.g., a DNA sequence aslarge as 3000 kb), that contains telomeric, centromeric, and origin ofreplication (replication origin) sequences.

A vector or plasmid of the present invention also encompasses bacterialartificial chromosome (BAC), which refers to a DNA construct that can begenetically modified to contain a heterologous DNA sequence (e.g., a DNAsequence as large as 300 kb), that contains an origin of replicationsequence (Ori), and may contain one or more helicases (e.g., parA, parB,and parC).

Examples of plasmids using Escherichia coli as their host includepBR322, pUC18, pUC19, pUC118, pVC119, pSP64, pSP65, pTZ-18R/-18U,pTZ-19R/-19U, pGEM-3, pGEM-4, pGEM-3Z, pGEM-4Z, pGEM-5Zf(−), andpBluescript KSTM (Stratagene). Examples of plasmids suitable forexpression in Escherichia coli include pAS, pKK223 (Pharmacia), pMC1403,pMC931, and pKC30.

Polynucleotides encoding the helper proteins and the POI may berecombined in to the host cell by ligating the relevant genes each intoone vector. It is possible to construct single vectors carrying thegenes, or two separate vectors, one to carry the helper protein genesand the other one the POI genes. These genes can be integrated into thehost cell genome by transforming the host cell using such vector orvectors. In some embodiments, the genes encoding the POI is integratedin the genome and the gene encoding the helper protein is integrated ina plasmid or vector. In some embodiments, the genes encoding the helperprotein is integrated in the genome and the gene encoding the POI isintegrated in a plasmid or vector. In some embodiments, the genesencoding the POI and the helper protein are integrated in the genome. Insome embodiments, the gene encoding the POI and the helper protein isintegrated in a plasmid or vector. If multiple genes encoding the POIare used, some genes encoding the POI are integrated in the genome whileothers are integrated in the same or different plasmids or vectors. Ifmultiple genes encoding the helper proteins are used, some of the genesencoding the helper protein are integrated in the genome while othersare integrated in the same or different plasmids or vectors.

Overexpression of the endogenous polypeptide in the recombinantmicrobial cell can be achieved by modifying expression controlsequences. Expression control sequences are known in the art andinclude, for example, promoters, enhancers, polyadenylation signals,transcription terminators, internal ribosome entry sites (IRES), and thelike, that provide for the expression of the polynucleotide sequence ina host cell. Expression control sequences interact specifically withcellular proteins involved in transcription (Maniatis et al., Science,236: 1237-1245 (1987)). Exemplary expression control sequences aredescribed in, for example, Goeddel, Gene Expression Technology: Methodsin Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

In a preferred embodiment, the overexpression is achieved by using anenhancer to express the helper protein. Transcriptional enhancers arerelatively orientation and position independent, having been found 5′and 3′ to the transcription unit, within an intron, as well as withinthe coding sequence itself. The enhancer may be spliced into theexpression vector at a position 5′ or 3′ to the coding sequence, but ispreferably located at a site 5′ from the promoter. Most yeast genescontain only one UAS, which generally lies within a few hundred basepairs of the cap site and most yeast enhancers (UASs) cannot functionwhen located 3′ of the promoter, but enhancers in higher eukaryotes canfunction both 5′ and 3′ of the promoter.

Many enhancer sequences are now known from mammalian genes (globin, RSV,SV40, EMC, elastase, albumin, a-fetoprotein and insulin). One may alsouse an enhancer from a eukaryotic cell virus, such as the SV40 lateenhancer, the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. Yeast enhancers, also called upstream activating sequences(UASs), such as the UAS/Gal system from Saccharomyces cerevisiae, can beadvantageously used with yeast promoters (described in European PatentNo. 0317254 and Rudoni et al., The International Journal of Biochemistryand Cell Biology, (2000), 32(2):215-224).

Overexpression of the endogenous polypeptide in the recombinant cell canbe achieved by modifying transcriptional and translational regulatorysequences, including, for example, promoters, enhancers, polyadenylationsignals, transcription terminators, internal ribosome entry sites(IRES), and the like, that provide for the expression of thepolynucleotide sequence in a host cell. Such sequences interactspecifically with cellular proteins involved in transcription (Maniatiset al., Science, 236: 1237-1245 (1987)). Exemplary sequences aredescribed in, for example, Goeddel, Gene Expression Technology: Methodsin Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990). Thepolynucleotides encoding the POI and/or helper proteins are preferablyoperably linked to transcriptional and translational regulatorysequences that provide for expression in the host cells. The term“transcriptional regulatory sequences” as used herein refers tonucleotide sequences that are associated with a gene nucleic acidsequence and which regulate the transcription of the gene. The term“translational regulatory sequences” as used herein refers to nucleotidesequences that are associated with a gene nucleic acid sequence andwhich regulate the translation of the gene. Transcriptional and/ortranslational regulatory sequences can either be located in plasmids orvectors or integrated in the chromosome of the host cell.Transcriptional and/or translational regulatory sequences are located inthe same nucleic acid molecule of the gene which it regulates.

For example, overexpression of the endogenous helper protein in therecombinant cell can be achieved by modifying the promoters, forexample, by replacing the endogenous promoter which is operably linkedto the helper protein with another stronger promoter in order to reachhigh expression levels. Such promoter may be inductive or constitutive.Modification of endogenous promoter may be performed by mutation orhomologous recombination using methods known in the art.

The overexpression of the polynucleotide encoding the helper proteins,can be achieved by other methods known in the art, for example bygenetically modifying their endogenous regulatory regions, as describedby Marx et al., 2008 (Marx, H., Mattanovich, D. and Sauer, M. MicrobCell Fact 7 (2008): 23), and Pan et al., 2011 (Pan et al., FEMS YeastRes. (2011) May; (3):292-8.), such methods include, for example,integration of a recombinant promoter that increases expression of thehelper proteins. Transformation is described in Cregg et al. (1985) Mol.Cell. Biol. 5:3376-3385. A “recombinant” promoter is referred to withrespect to the sequence whose expression it drives. As used herein, arecombinant promoter means when the promoter is not a native promoter tothe given sequence, i.e., when the promoter is different from anaturally occurring promoter (the “native promoter”). The term“promoter” as used herein refers to a region that facilitates thetranscription of a particular gene. A promoter typically increases theamount of recombinant product expressed from a nucleotide sequence ascompared to the amount of the expressed recombinant product when nopromoter exists. A promoter from one organism can be utilized to enhancerecombinant product expression from a sequence that originates fromanother organism. The promoter can be integrated into a host cellchromosome by homologous recombination using methods known in the art(e.g. Datsenko et al, Proc. Natl. Acad. Sci. U.S.A., 97(12): 6640-6645(2000)). In addition, one promoter element can increase the amount ofproducts expressed for multiple sequences attached in tandem. Hence, onepromoter element can enhance the expression of one or more recombinantproducts.

Promoter activity may be assessed by its transcriptional efficiency.This may be determined directly by measurement of the amount of mRNAtranscription from the promoter, e.g. by Northern Blotting, quantitativePCR or indirectly by measurement of the amount of gene product expressedfrom the promoter.

The promoter could be an “inducible promoter” or “constitutivepromoter.” “Inducible promoter” refers to a promoter which can beinduced by the presence or absence of certain factors, and “constitutivepromoter” refers to an unregulated promoter that allows for continuoustranscription of its associated gene or genes.

In a preferred embodiment, both the nucleotide sequences encoding thehelper protein and the POI are driven by an inducible promoter. Inanother preferred embodiment, both the nucleotide sequences encoding thehelper protein and POI is driven by a constitutive promoter. In yetanother preferred embodiment, the nucleotide sequences encoding thehelper protein is driven by a constitutive promoter and the POI isdriven by an inducible promoter. In yet another preferred embodiment,the nucleotide sequences encoding the helper protein is driven by aninducible promoter and the POI is driven by a constitutive promoter. Asan example, the HP may be driven by a constitutive GAP promoter and thePOI may be driven by an inducible AOX1 promoter. In one embodiment, thenucleotide sequences encoding the helper protein and POI is driven bythe same promoter or similar promoters in terms of promoter activityand/or expression behaviour.

Many inducible promoters are known in the art. Many are described in areview by Gatz, Curr. Op. Biotech., 7: 168 (1996) (see also Gatz, Ann.Rev. Plant. Physiol. Plant Mol. Biol., 48:89 (1997)). Examples includetetracycline repressor system, Lac repressor system, copper-induciblesystems, salicylate-inducible systems (such as the PR1 a system),glucocorticoid-inducible (Aoyama et al., 1997), alcohol-induciblesystems, e.g., AOX promoters, and ecdysome-inducible systems. Alsoincluded are the benzene sulphonamide-inducible (U.S. Pat. No.5,364,780) and alcohol-inducible systems (WO 97/06269 and WO 97/06268)and glutathione 5-transferase promoters.

Suitable promoter sequences for use with yeast host cells are describedin Mattanovich et al., Methods Mol. Biol. (2012) 824:329-58 and includeglycolytic enzymes like triosephosphate isomerase (TPI),phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase(GAPDH or GAP) and variants thereof, lactase (LAC) and galactosidase(GAL), P. pastoris glucose-6-phosphate isomerase promoter (PPGI, the3-phosphoglycerate kinase promoter (PPGK), the glycerol aldehydephosphate dehydrogenase promoter (PGAP), translation elongation factorpromoter (PTEF), and the promoters of P. pastoris enolase 1 (PENO1),triose phosphate isomerase (PTPI), ribosomal subunit proteins (PRPS2,PRPS7, PRPS31, PRPL1), alcohol oxidase promoter (PAOX) or variantsthereof with modified characteristics, the formaldehyde dehydrogenasepromoter (PFLD), isocitrate lyase promoter (PICL), alpha-ketoisocaproatedecarboxylase promoter (PTHI), the promoters of heat shock proteinfamily members (PSSA1, PHSP90, PKAR2), 6-Phosphogluconate dehydrogenase(PGND1), phosphoglycerate mutase (PGPM1), transketolase (PTKL1),phosphatidylinositol synthase (PPIS1), ferro-O2-oxidoreductase (PFET3),high affinity iron permease (PFTR1), repressible alkaline phosphatase(PPHO8), N-myristoyl transferase (PNMT1), pheromone responsetranscription factor (PMCM1), ubiquitin (PUBI4), single-stranded DNAendonuclease (PRAD2), the promoter of the major ADP/ATP carrier of themitochondrial inner membrane (PPET9) (WO2008/128701) and the formatedehydrogenase (FMD) promoter. The GAP promoter, AOX promoter or apromoter derived from GAP or AOX promoter is particularly preferred. AOXpromoters can be induced by methanol and are repressed by e.g. glucose.

Further examples of suitable promoters include Saccharomyces cerevisiaeenolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1),Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase (PG K), and the maltase gene promoter (MAL).

Other useful promoters for yeast host cells are described by Romanos etal, 1992, Yeast 8:423-488.

Suitable promoter sequences for use with E. coli include T7 promoter, T5promoter, tryptophan (trp) promoter, lactose (lac) promoter,tryptophan/lactose (tac) promoter, lipoprotein (lpp) promoter, and Aphage PL promoter in plasmids.

The promoter which drives the expression of the polynucleotide encodingthe helper protein is preferably not the endogenous to the promoter ofthe helper gene. Preferably, a recombinant promoter is used instead ofthe endogenous promoter of the helper protein gene. A “recombinant”promoter is referred to with respect to the sequence whose expression itdrives. As used herein, a recombinant promoter means when the promoteris not a native promoter to the given sequence, i.e., when the promoteris different from a naturally occurring promoter (the “nativepromoter”). Such a promoter is sometimes also referred to herein asheterologous promoter.

In a preferred embodiment, the overexpression is achieved by using anenhancer to enhance the promoter activity which drives the expression ofthe helper protein. Transcriptional enhancers are relatively orientationand position independent, having been found 5′ and 3′ to thetranscription unit, within an intron, as well as within the codingsequence itself. The enhancer may be spliced into the expression vectorat a position 5′ or 3′ to the coding sequence, but is preferably locatedat a site 5′ from the promoter. Most yeast genes contain only one UAS,which generally lies within a few hundred base pairs of the cap site andmost yeast enhancers (UASs) cannot function when located 3′ of thepromoter, but enhancers in higher eukaryotes can function both 5′ and 3′of the promoter.

Many enhancer sequences are now known from mammalian genes (globin, RSV,SV40, EMC, elastase, albumin, a-fetoprotein and insulin). One may alsouse an enhancer from a eukaryotic cell virus, such as the SV40 lateenhancer, the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. Yeast enhancers, also called upstream activating sequences(UASs), such as the UAS/Gal system from Saccharomyces cerevisiae, can beadvantageously used with yeast promoters (described in European PatentNo. 0317254 and Rudoni et al., The International Journal of Biochemistryand Cell Biology, (2000), 32(2):215-224).

It is preferred that the present host cell underexpressing the KOprotein(s) would coexpress the protein of interest and a helper proteinhaving an amino acid sequence having at least 30% sequence identity toan amino acid sequence as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,or 162.

Preferably, the host cell can be engineered to overexpress 2, 3, 4, 5,6, 7, 8 or more of helper proteins selected from SEQ ID NO: 1, 2, 3, 4,5, 6, 7, 8, 9, or 162 or functional homologues thereof. In a preferredembodiment, the host cell can be engineered to (1) underexpress 1, 2, 3or more of KO proteins KO1, KO2, KO3 or functional homologues thereofand (2) overexpress 1, 2, 3, 4, 5, 6, 7, 8, 9 or more of helper proteinsHP1, HP2, HP3, HP4, HP5, HP6, HP7, HP8, HP9, HP10 or functionalhomologues thereof.

However, a specific combination of helper proteins HP3 (SEQ ID NO: 2) ora functional homologue and HP1 (SEQ ID NO. 4) or a functional homologue,or helper proteins HP10 (SEQ ID NO: 162) or a functional homologue andHP3 (SEQ ID NO: 2) or a functional homologue, is a preferred embodimentof the host cells of the present invention that are preferably appliedin the methods and uses of the present invention.

Also, a specific combination of helper protein HP2 (SEQ ID NO: 1) or afunctional homologue and HP3 (SEQ ID NO: 2) or a functional homologueand KO protein KO1 (SEQ ID NO: 10), or helper protein HP2 (SEQ ID NO: 1)or a functional homologue and HP1 (SEQ ID NO: 4) or a functionalhomologue and KO protein KO1 (SEQ ID NO: 10), is a preferred embodimentof the host cells of the present invention that are preferably appliedin the methods and uses of the present invention.

Host cells being engineered to reflect such a combination are envisagedin a preferred embodiment. These host cells are preferably applied inthe methods and uses described herein. “Reflected” means that theskilled person knows in accordance with the teaching of the presentinvention that a helper protein is overexpressed, while a KO protein isunderexpressed.

It is preferred that the present host cell for manufacturing a proteinof interest is engineered to underexpress at least one polynucleotideencoding a KO protein having an amino acid sequence as shown in SEQ IDNO: 10, 11, 12 or a functional homologue thereof and furtheroverexpresses at least one polynucleotide encoding a helper proteinhaving an amino acid sequence as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6,7, 8, 9, or 162 or a functional homologue thereof, wherein thefunctional homologue of the helper protein has at least 30%, such as atleast 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or100%, sequence identity to an amino acid sequence as shown in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, or 162.

In a preferred embodiment, the underexpression of at least onepolynucleotide encoding a KO protein, in concert with the overexpressionof at least one polynucleotide encoding a helper protein, may increasethe yield of the protein of interest, for example SDZ-Fab (SEQ ID NO: 25and 26) or HyHEL-Fab (SEQ ID NO: 29 and 20) in the host cell by at least1%, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or at least200% or more, compared to the host cell prior to the engineering tounderexpress the polynucleotide encoding the KO protein.

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,constructs, and reagents described, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention, which will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are for the purpose of describing anddisclosing, for example, the cell lines, constructs, and methodologiesthat are described in the publications, which might be used inconnection with the presently described invention. The publicationsdiscussed above and throughout the text are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention and defined in the claims.Efforts have been made to ensure accuracy with respect to the numbersused (e.g. amounts, temperature, concentrations, etc.) but someexperimental errors and deviations should be allowed for. Unlessotherwise indicated, parts are parts by weight, molecular weight isaverage molecular weight, temperature is in degrees centigrade; andpressure is at or near atmospheric.

EXAMPLES

The below examples will demonstrate that the newly identified gene(s)increases the titer (product per volume in mg/L) and the yield (productper biomass in mg/g biomass measured as dry cell weight or wet cellweight), respectively, of recombinant proteins upon its/theirunderexpression. As an example, the yield of recombinant antibody Fabfragments and recombinant enzymes in the yeast Pichia pastoris areincreased. The positive effect was shown in shaking cultures (conductedin shake flasks or deep well plates) and in lab scale fed-batchcultivations.

Example 1 Generation of P. pastoris Production Strains a) Constructionof P. pastoris Strains Secreting Antibody Fab Fragment HyHEL

P. pastoris CBS7435 (CBS, genome sequenced by Küberl et al. 2011)mut^(s) variant was used as host strain. The pPM2d_pGAP and pPM2d_pAOXexpression vectors are derivatives of the pPuzzle_ZeoR vector backbonedescribed in WO2008/128701A2, consisting of the pUC19 bacterial originof replication and the Zeocin antibiotic resistance cassette. Expressionof the heterologous gene is mediated by the P. pastorisglyceraldehyde-3-phosphate dehydrogenase (GAP) promoter or alcoholoxidase (AOX) promoter, respectively, and the S. cerevisiae CYC1transcription terminator. The light chain (LC) and the heavy chain (HC)of the antibody Fab fragment HyHEL (FIG. 2) were amplified from vectorDNA template (carrying the gene of interests with N-terminal S.cerevisiae alpha mating factor signal leader sequence) using the primersfor HyHEL-HC and HyHEL-LC in Table 3, and each ligated into both vectorspPM2d_pGAP and pPM2d_pAOX digested with SbfI and SfiI. The LC fragmentswere ligated into variants of pPM2d_pGAP and pPM2d_pAOX, where onerestriction enzyme site in the promoter region was exchanged for anotherto allow subsequent linearization (NdeI instead of AwlI in pPM2d_pGAP,Bsu36I instead of Bpu1102I in pPM2d_pAOX), the HC fragments were ligatedinto the unmodified versions of the vectors. After sequence verificationof LC and HC, the expression cassettes for both chains were combinedonto one vector by using the compatible restriction enzymes MreI andAgeI.

Plasmids were linearized using NdeI restriction enzyme (for pPM2d_pGAP)or Bsu36I restriction enzyme (for pPM2d_pAOX), respectively, prior toelectroporation (using a standard transformation protocol described inGasser et al. 2013. Future Microbiol. 8(2):191-208) into P. pastoris.Selection of positive transformants was performed on YPD plates (perliter: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar-agar)containing 50 μg/mL of Zeocin. Colony PCR was used to ensure thepresence of the transformed plasmid. Therefore, genomic DNA was obtainedby cooking and freezing of P. pastoris colonies for 5 minutes each anddirectly applied for PCR with the appropriate primers.

b) Construction of a P. pastoris Strain Secreting Antibody Fab FragmentSDZ

The light chain (LC) and the heavy chain (HC) of the antibody Fabfragment SDZ (FIG. 2) were amplified from vector DNA template (carryingthe gene of interests with N-terminal alpha mating factor signal leadersequence) using the primers for SDZ-HC and SDZ-LC in Table 3, and eachligated into pPM2d_pAOX or the variant of pPM2d_pAOX with the Bsu36Irestriction site, respectively, each digested with SbfI and SfiI. Aftersequence verification of LC and HC, the expression cassettes for bothchains were combined onto one vector by using the compatible restrictionenzymes MreI and AgeI.

Plasmids were linearized using Bsu36I restriction enzyme prior toelectroporation (using a standard transformation protocol described inGasser et al. 2013. Future Microbiol. 8(2):191-208) into P. pastoris.Selection of positive transformants was performed on YPD plates (perliter: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar-agar)containing 50 μg/mL of Zeocin. Colony PCR was used to ensure thepresence of the transformed plasmid. Therefore, genomic DNA was obtainedby cooking and freezing of P. pastoris colonies for 5 minutes each anddirectly applied for PCR with the appropriate primers.

Table 3 shows oligonucleotide primers for PCR amplification of HyHEL LCand HC as well as SDZ LC and HC (Alpha-mating factor_forward is theforward primer for amplification of all Fab chains).

TABLE 3 Restriction site Primer attached sequence Alpha-mating factor_SbfI ACTACCTGCAGGCGAAACGATGAGATTCCCATC forward* SEQ ID NO: 33HyHEL-HC backward SfiI TCATGGCCGAGGCGGCCCTATTACTTGTCACAGG ACTTTGGCTCSEQ ID NO: 34 HyHEL-LC backward SfiI CTATGGCCGAGGCGGCCCTATTAACACTCACCTCTGTTG SEQ ID NO: 35 SDZ-HC back SfiI TATCGGCCGAGGCGGCCCTATTACTTACCTGGGGACAAG SEQ ID NO: 36 SDZ-LC back SfiI CTATGGCCGAGGCGGCCCTATTAACACTCACCTCTGTTG SEQ ID NO: 37

Example 2 Chemostat Cultivation

Cultivations were performed with 1.4 L DASGIP reactors (Eppendorf,Germany) with a maximum working volume of 1.0 L.

Following media were used:

PTM₁ trace salts stock solution (per litre) contains: 6.0 g CuSO₄.5H₂O,0.08 g NaI, 3.36 g MnSO₄. H₂O, 0.2 g Na₂MoO₄.2H₂O, 0.02 g H₃BO₃, 0.82 gCoCl₂, 20.0 g ZnCl₂, 65.0 g FeSO₄.7H₂O, and 5.0 mL H₂SO₄(95%-98%).

Glycerol Batch medium (per litre) contains: 2 g Citric acid monohydrate(C₆H₈O₇.H₂O), 39.2 g Glycerol, 20.8 g NH₄H₂PO₄, 0.5 g MgSO₄.7H₂O, 1.6 gKCl, 0.022 g CaCl₂.2H₂O, 0.8 mg biotin and 4.6 mL PTM1 trace salts stocksolution. HCl was added to set the pH to 5.

Glucose Chemostat medium (per litre) contains: 2.5 g Citric acidmonohydrate (C₆H₈O₇.H₂O), 55.0 g glucose monohydrate, 21.8 g NH₄H₂PO₄,1.0 g MgSO₄.7H₂O, 2.5 g KCl, 0.04 g CaCl₂.2H₂O, 4.0 mg biotin and 2.43mL PTM1 trace salts stock solution. HCl was added to set the pH to 5.

Methanol/Glycerol Chemostat medium (per litre) contains: 2.5 g Citricacid monohydrate (C₆H₈O₇.H₂O), 8.5 g methanol, 50.0 g glycerol, 21.8 gNH₄H₂PO₄, 1.0 g MgSO₄.7H₂O, 2.5 g KCl, 0.04 g CaCl₂.2H₂O, 4.0 mg biotinand 2.43 mL PTM1 trace salts stock solution. HCl was added to set the pHto 5.

The dissolved oxygen was controlled at DO=20% with stirrer speed(400-1200 rpm) and aeration rate (12-72 standard Liter/hour (sL/h)) air,the temperature was controlled at 25° C. and the pH setpoint of 5 wascontrolled with addition of NH₄OH (25%). Foaming was controlled byaddition of antifoam agent (5% Glanapon 2000) on demand.

To start the cultivation, 0.4 L batch medium was sterile filtered andtransferred into the fermenter under a sterile work bench and wasinoculated (from a P. pastoris overnight pre-culture in YPG containing50 μg/mL Zeocin, 180 rpm, 28° C.) with a starting optical density(OD₆₀₀) of 1. The batch phase of approximately 24 h reached a drybiomass concentration of approximately 20 g/L, it was followed by aconstant feed with chemostat medium at 40 mL/h (for p=D=0.1/h) withsimultaneous constant removal of culture to keep the total volumeconstant. A dry biomass concentration of approximately 25 g/L wasreached after 7 residence times (70 hours) when samples for microarrayexperiments were taken and the cultivation was terminated. Thischemostat cultivation was performed three times with each productionstrain (CBS7435 pGAP HyHEL-Fab using glucose chemostat medium andCBS7435mut^(s) pAOX HyHEL-Fab using methanol/glycerol chemostat medium)and non-producing wildtype control strain (CBS7435 pGAP control andCBS7435mut^(s) pAOX control, respectively) to obtain the biologicalreplicates necessary for reliable microarray analysis.

Samples were taken after approximately 70 hours in steady stateconditions of the chemostat. Routine sampling as determination ofoptical density or yeast dry mass, qualitative microscopic inspectionand cell viability analysis was done alongside during each cultivation.For microarray analysis, samples were taken and treated as follows: Foroptimal quenching, 9 mL cell culture broth was immediately mixed with4.5 mL of ice cold 5% phenol (Sigma) solution (in Ethanol abs.), andaliquoted. Each 2 mL were centrifuged (13,200 rpm for 1 minute) inpre-cooled collection tubes (GE healthcare, NJ), supernatant was removedcompletely and the tubes were stored at −80° C. until RNA isolation.

Example 3 Microarrays & Data Evaluation for Transcriptomic Experimentsa) RNA isolation and sample preparation for microarray hybridization

The RNA was isolated from chemostat sample cells using TRI reagentaccording to the supplier's instructions (Ambion, US). The cell pelletswere resuspended in TRI reagent and homogenized with glass beads using aFastPrep 24 (M.P. Biomedicals, CA) at 5 m s⁻¹ for 40 seconds. Afteraddition of chloroform, the samples were centrifuged and the total RNAwas precipitated from the aqueous phase by adding isopropanol. Thepellet was washed with 70% ethanol, dried and re-suspended in RNAse freewater. RNA concentrations were determined by measuring OD₂₆₀ using aNanodrop 1000 spectrophotometer (NanoDrop products, DE). Remaining DNAfrom the samples was removed using the DNA free Kit (Ambion, CA). Samplevolume equal to 10 μg RNA was diluted to 50 μL in RNAse free water, thenDNAse buffer I and rDNAse I were added and incubated at 37° C. for 30minutes. After addition of DNAse Inactivation Reagent, the sample wascentrifuged and the supernatant was transferred into a fresh tube. RNAconcentrations were determined again like described above. Additionally,RNA integrity was analyzed using RNA nano chips (Agilent). To monitorthe microarray workflow from amplification and labelling tohybridisation of the samples, the Spike In Kit (Agilent, Product Nr.:5188-5279) was used as positive control. It contains 10 differentpolyadenylated transcripts from an adenovirus, which are amplified,labelled and cohybridised together with the own RNA samples. The sampleswere labelled with Cy 3 and Cy 5 using the Quick Amp Labelling Kit(Agilent, Prod. Nr. 5190-0444). Therefore 500 ng of purified sample RNAwere diluted in 8.3 μL RNAse free water, 2 μL Spike A or B, and 1.2 μLT7 promoter primer were added. The mixture was denatured for 10 minutesat 65° C. and kept on ice for 5 minutes. Then 8.5 μL cDNA mastermix (persample: 4 μL 5× first strand buffer, 2 μL 0.1 M DTT, 1 μL 10 mM dNTPmix, 1 μL MMLV-RT, 0.5 μL RNAse out) were added, incubated at 40° C. for2 hours, then transferred to 65° C. for 15 minutes and put on ice for 5minutes. The transcription mastermix (per sample: 15.3 μL nuclease freewater, 20 μL transcription buffer, 6 μL 0.1 M DTT, 6.4 μL 50% PEG, 0.5μL RNAse Inhibitor, 0.6 μL inorg. phosphatase, 0.8 μL T7 RNA Polymerase,2.4 μL Cyanin 3 or Cyanin 5) was prepared and added to each tube andincubated at 40° C. for 2 hours. In order to purify the obtainedlabelled cRNA, the RNeasy Mini Kit (Qiagen, Cat. No. 74104) was used.Samples were stored at −80° C. Quantification of the cRNA concentrationand labelling efficiency was done at the Nanodrop spectrophotometer.

b) Microarray Analysis

The Gene Expression Hybridisation Kit (Agilent, Cat. No. 5188-5242) wasused for hybridisation of the labelled sample cRNAs. For the preparationof the hybridisation samples each 300 ng cRNA (Cy3 and Cy 5) and 6 μL10-fold blocking agent were diluted with nuclease free water to a finalvolume of 24 μL. After addition of 1 μL 25-fold fragmentation buffer,the mixture was incubated at 60° C. for 30 minutes. Then 25 μL GExHybridisation Buffer HI-RPM was added to stop the reaction. Aftercentrifugation for one minute with 13,200 rpm, the sample was chilled onice and used for hybridisation immediately. In-house designed P.pastoris specific oligonucleotide arrays (AMAD-ID: 034821, 8×15K customarrays, Agilent) were used. Microarray hybridisation was done accordingto the Microarray Hybridisation Chamber User Guide (Agilent G2534A).First, the gasket slide was uncovered and put onto the chamber base,Agilent label facing up. The sample (40 μL per array) was loaded in themiddle of each of the eight squares. Then the microarray slide wascarefully put onto the gasket slide (Agilent label facing down) and thechamber cover was placed on and fixed with the clamp. All samples werehybridized against a reference pool sample in a dye-swap manner. Thepool RNA sample had been generated by combining RNA from a variety ofcultivations in equal amounts. Hybridisation was done in thehybridisation oven for 17 hours at 65° C. Before scanning, themicroarray chip was washed. Therefore, the chamber was dismantled, andthe sandwich slides were detached from each other while submerged inwash buffer 1. The microarray was directly transferred into another dishwith wash buffer 1, washed for 1 minute, transferred into wash buffer 2(temperature at least 30° C.) and washed for another minute. Afterdrying of the microarray slide by touching the slide edge with a tissue,it was put into the slide holder (Agilent label facing up). The slideholder was put into the carousel and scanning was started.

c) Data Acquisition and Statistical Evaluation of Microarray Data

Images were scanned at a resolution of 50 nm with a G2565AA Microarrayscanner (Agilent) and were imported into the Agilent Feature Extraction9.5 software. Agilent Feature Extraction 9.5 was used for thequantification of the spot intensities. The raw mean spot intensity datawas then imported into the open source software R for furthernormalisation and data analysis.

The intensity data were subjected to normalization (no backgroundcorrection, the within slide normalization method Loess and the betweenslide normalization method Aquantile was used), before the differentialexpression values were calculated. The p-values associated with thedifferential expression values were calculated using a linear model fit(limma R package), subsequently they were adjusted for multiple testingusing the method of Benjamini and Yekutieli (BY method of limma Rpackage). Log 2 fold changes were calculated for HyHEL-Fab producingstrains compared to their respective control.

The microarray data was browsed for entries with significant (adjustedp-value <0.05) difference in expression levels (fold change >1.5)between the chemostat triplicates of CBS7435 producing HyHEL-Fab and itsnon-producing host control.

Table 4 shows up-regulated genes from microarray analysis of HyHEL-Fabproducing P. pastoris.

TABLE 4 Transcript level Transcript level fold fold change in Geneidentifier change in CBS7435 CBS7435 P. pastoris Microarray pPM2d_pGAPHyHEL pPM2d_pAOX CBS7435 probe name vs. control HyHEL vs. controlPAS_chr4_0822 Pipas_chr4_0822 1.20 8.27 PP7435_Chr4-1007 Pipas_chr4_00092.68 5.56 PP7435_Chr3-0183 Pipas_chr3_0987 2.17 5.47 PP7435_Chr1-1225Pipas_chr1-4_0431 2.48 4.76 PP7435_Chr4-0976 PIPA00444 2.27 4.22PP7435_Chr2-0351 Pipas_chr2-2_0331 1.74 4.10 PP7435_Chr1-0941Pipas_chr1-4_0167 0.93 3.12 PP7435_Chr3-0933 Pipas_chr3_0288 2.06 3.11PAS_chr3_0401 Pipas_chr3_0401 2.59 2.92 PP7435_Chr1-1232Pipas_chr1-4_0681 1.57 2.79 PP7435_Chr4-0294 Pipas_chr4_0673 2.11 2.70PP7435_Chr1-0667 Pipas_chr1-1_0348 2.02 2.69 PP7435_Chr3-0607Pipas_chr3_0598 2.02 2.59 PP7435_Chr2-0722 Pipas_chr2-1_0566 1.13 2.50PP7435_Chr2-0842 Pipas_chr2-1_0454 4.16 2.43 PAS_chr3_0821Pipas_chr3_0821 1.40 2.18 PP7435_Chr2-0501 Pipas_chr2-1_0887 2.69 2.18PP7435_Chr2-0220 Pipas_chr2-2_0210 1.27 2.17 PP7435_Chr3-0278Pipas_chr3_0895 1.06 2.09 PP7435_Chr4-0108 Pipas_chr4_0843 1.80 2.04PP7435_Chr2-1019 Pipas_chr2-1_0287 1.52 2.04 PP7435_Chr3-1062Pipas_chr3_0170 1.75 1.96 PP7435_Chr4-0448 Pipas_chr4_0972 2.31 1.95PP7435_Chr3-0837 Pipas_chr3_0377 1.38 1.87 PP7435_Chr1-0176Pipas_chr1-3_0174 1.70 1.84 PP7435_Chr3-0156 Pipas_chr3_1014 1.30 1.84PP7435_Chr4-0582 Pipas_chr4_0403 1.34 1.82 PP7435_Chr2-0638Pipas_chr2-1_0642 2.35 1.78 PP7435_Chr3-0639 Pipas_chr3_0567 1.70 1.73PP7435_Chr2-0866 Pipas_chr2-1_0433 1.36 1.71 PP7435_Chr2-0028Pipas_chr2-2_0031 1.70 1.71 PP7435_Chr1-1009 Pipas_chr1-4_0229 4.09 1.70PP7435_Chr2-0848 Pipas_chr2-1_0448 1.39 1.70 PP7435_Chr1-0470Pipas_chr1-1_0160 1.39 1.69 PP7435_Chr1-0077 Pipas_chr1-3_0080 1.31 1.68PP7435_Chr1-1220 Pipas_chr1-4_0428 1.49 1.68 PP7435_Chr1-0571Pipas_chr1-1_0259 2.18 1.68 PP7435_Chr1-0204 Pipas_chr1-3_0202 0.71 1.67PP7435_Chr4-0182 Pipas_chr4_0775 1.22 1.65 PP7435_Chr4-0320Pipas_chr4_0650 1.26 1.65 PP7435_Chr3-0548 Pipas_chr3_0652 2.29 1.62PP7435_Chr2-1300 Pipas_chr2-1_0002 3.71 1.62 PP7435_Chr1-1077Pipas_chr1-4_0294 1.46 1.61 PP7435_Chr4-0699 Pipas_chr4_0299 1.43 1.61PP7435_Chr2-0729 Pipas_chr2-1_0560 1.12 1.56 PP7435_Chr4-0923Pipas_chr4_0093 0.94 1.53 PP7435_Chr1-0592 Pipas_chr1-1_0276 1.59 1.52

Example 4 Generation of Strains Overexpressing Identified Genes

For the investigation of positive effects on Fab secretion, theidentified genes were overexpressed in two different Fab producingstrains: CBS7435 pPM2d_pAOX HyHEL, which was the source of themicroarray data (see Example 3) and CBS7435 pPM2d_pAOX SDZ (generationsee Example 1).

a) Amplification and Cloning of the Identified Potential SecretionHelper Genes into pPM2aK21 Expression Vectors

The genes identified in Example 3 were amplified by PCR (PhusionPolymerase, New England Biolabs) from start to stop codon using theprimers shown in Table 5. The sequences were cloned into the MCS of thepPM2a expression vector with the two restriction enzymes SbtI and SfiI.pPMKaK21 is a derivative of pPM2d (described in Example 1a), consistingof an AOX terminator sequence (for integration into the native AOXterminator locus), an origin of replication for E. coli (pUC19), anantibiotic resistance cassette (kanMX conferring resistance to Kanamycinand G418) for selection in E. coli and yeast, an expression cassette forthe gene of interest (G01) consisting of a GAP promoter, a multiplecloning site (MCS) and the S. cerevisiae CYC1 transcription terminator.Gene sequences were verified by Sanger sequencing.

TABLE 5 Gene identifier (ORF name CBS7435)Forward primer (SbfI attached) Backward primer (SfiI attached)PAS_chr4_0822 CTTGCCTGCAGGATGCTAACGGCCAG GATCGGCCGAGGCGGCCTCAGCAG TTGGTCTATTCCCACCAGAATC SEQ ID NO: 38 SEQ ID NO: 39 PP7435_Chr4-CTTGCCTGCAGGATGTCAGTTCATTTC GATCGGCCGAGGCGGCCTCATATAA 1007 GTTATAGCAGCAAGGTTTATCATAATTCTCATCCTCA SEQ ID NO: 40 G SEQ ID NO: 41 PP7435_Chr3-GAAACCTGCAGGATGTCTGAATTTGTT GATCGGCCGAGGCGGCCTTAGGCG 0183GCTAAAATTAACATTC GTTGGAACGTTC SEQ ID NO: 42 SEQ ID NO: 43 PP7435_Chr1-GAAACCTGCAGGATGTCTCATCTATTA GATCGGCCGAGGCGGCCTCATGGC 1225 CTGCGTGACAGCCCGGCATATCTAG SEQ ID NO: 44 SEQ ID NO: 45 PP7435_Chr4-GACACCTGCAGGATGTCTGAATCCTC GATCGGCCGAGGCGGCCCTAGATAC 0976CAGTATCTCTCTAGTTG ATCCCAAAAGTGCACCG SEQ ID NO: 46 SEQ ID NO: 47PP7435_Chr2- GATACCTGCAGGATGATCCTGGGTTC GATCGGCCGAGGCGGCCCTAAAAGT 0351AGTTTGGG TTGCTGCAGCATTTGAAG SEQ ID NO: 48 SEQ ID NO: 49 PP7435_Chr1-CTTGCCTGCAGGATGGGTTGCTTTAG GATCGGCCGAGGCGGCCCTATTTGT 0941 ATTTTGTCTGGATACGTGCTGTGGAGCC SEQ ID NO: 50 SEQ ID NO: 51 PP7435_Chr3-CTTGCCTGCAGGATGTTAAACAAGCTG GATCGGCCGAGGCGGCCTTAGCTG 0933TTCATTGCAATACTC GCAAGGGTAATTGTCTC SEQ ID NO: 52 SEQ ID NO: 53PAS_chr3_0401 GACACCTGCAGGATGGCTCCTCAAAC GATCGGCCGAGGCGGCCTCAAAAAAACCAAGG ACAATCTCAAAATCTCCAG SEQ ID NO: 54 SEQ ID NO: 55 PP7435_Chr1-GTACCCTGCAGGATGACCAAGGAAAA GATCGGCCGAGGCGGCCTTATTTTTT 1232 TGAAGCCCTCCAATTCAGCCAG SEQ ID NO: 56 SEQ ID NO: 57 PP7435_Chr4-GAAACCTGCAGGATGCTGTTGTCACAT GATCGGCCGAGGCGGCCTTAAGATT 0294 ACCATGATACTTCGCTTCTTTTTGAGATTGG SEQ ID NO: 58 SEQ ID NO: 59 PP7435_Chr1-GAAACCTGCAGGATGACGGACTATGT GATCGGCCGAGGCGGCCTCATAATC 0667 CACTTCTAAGCGTCCCTCCAGGGG SEQ ID NO: 60 SEQ ID NO: 61 PP7435_Chr3-GAAACCTGCAGGATGTCGTCTGATGC GATCGGCCGAGGCGGCCTCAAATAA 0607 TGTGGAGTGCTACATTTGCGTTTCTTTC SEQ ID NO: 62 SEQ ID NO: 63 PP7435_Chr2-CTTGCCTGCAGGATGTCTTATACGTCG GATCGGCCGAGGCGGCCTTACGTGT 0722 GACAACAAAGAGATCCGCTTCCTCTGTAC SEQ ID NO: 64 SEQ ID NO: 65 PP7435_Chr2-GATACCTGCAGGATGAACTTGTACCTA GATCGGCCGAGGCGGCCTTAGAACC 0842ATTACATTACTATTCGC CACATTGATTTGGATACTG SEQ ID NO: 66 SEQ ID NO: 67PAS_chr3_0821 GAAACCTGCAGGATGTCGTTATCAACC GATCGGCCGAGGCGGCCTCAGACTCTTTCTAGGCG TACTCATCATTTTGTCTTCCTC SEQ ID NO: 68 SEQ ID NO: 69PP7435_Chr2- GAAACCTGCAGGATGATGTACAGGAA GATCGGCCGAGGCGGCCCTAACACT 0501CTTAATAATTGCTACTGC CTATGAGGTCTACAATGTCCAAC SEQ ID NO: 70 SEQ ID NO: 71PP7435_Chr2- CATGCCTGCAGGATGTCTACAGCAATT GATCGGCCGAGGCGGCCTTAGTTGA 0220CCAGGAGGAC TCAACTTTCCTGTCAGCTTAG SEQ ID NO: 72 SEQ ID NO: 73PP7435_Chr3- GACACCTGCAGGATGAGTGGTGACCA GATCGGCCGAGGCGGCCTTACTGTG 0278TAAGAGCTTTACG TACCATACCGATCCAATCC SEQ ID NO: 74 SEQ ID NO: 75PP7435_Chr4- CTTGCCTGCAGGATGACTAACTGGAA GATCGGCCGAGGCGGCCTTAGTTCT 0108AGCGATATTGACTCC CTTCTTCACCTTGAAATTTTAGGC SEQ ID NO: 76 SEQ ID NO: 77PP7435_Chr2- GCAACCTGCAGGATGTCTTATCGCCC GATCGGCCGAGGCGGCCTCAATAGA 1019TCAGTTTCAAC TCTTTTTCTTTTCATCAAAACTCAAC SEQ ID NO: 78 SEQ ID NO: 79PP7435_Chr3- GATACCTGCAGGATGGTGGTGCACAA GATCGGCCGAGGCGGCCCTAGGTA 1062CCCTAATAAC CTATGCTGAACATCCTGAGTATGAG SEQ ID NO: 80 SEQ ID NO: 81PP7435_Chr4- GAAACCTGCAGGATGAGATTTTCTAAC GATCGGCCGAGGCGGCCTTACAAGG 0448GTCGTTTTAACTGC CAAAGACTCCGAAAGTG SEQ ID NO: 82 SEQ ID NO: 83PP7435_Chr3- GACACCTGCAGGATGACTGTGCCTGA GATCGGCCGAGGCGGCCTCAGGCC 0837TCTGAAAGAAAC AGCGCAACG SEQ ID NO: 84 SEQ ID NO: 85 PP7435_Chr1-CTTGCCTGCAGGATGAAGATATGGCT GATCGGCCGAGGCGGCCTCACAATT 0176GGTACTTCTTTTAGTTTTTG CGTCTCTAATTTGTTGCG SEQ ID NO: 86 SEQ ID NO: 87PP7435_Chr3- CTTGCCTGCAGGATGGAGCAGGTTCC GATCGGCCGAGGCGGCCTTATTCAT 0156AGTCG CATAAACTTCTTCTATGGTGGC SEQ ID NO: 88 SEQ ID NO: 89 PP7435_Chr4-CTTGCCTGCAGGATGGATCCTTTTTCA GATCGGCCGAGGCGGCCCTACTTTG 0582 ATTCTTCTCACGAGACAGATCTTCCACCTTAAC SEQ ID NO: 90 SEQ ID NO: 91 PP7435_Chr2-GAAACCTGCAGGATGACCAGTCAAGG GATCGGCCGAGGCGGCCCTATATGC 0638 ATTTTTGGATCTATCAACCATCTCCATCAAATAAC SEQ ID NO: 92 SEQ ID NO: 93 PP7435_Chr3-GACACCTGCAGGATGACTCCCCGTTC GATCGGCCGAGGCGGCCCTACTCAA 0639 TCATATTTTCAGAACTTAGACAAAGCAGCTTTCTC SEQ ID NO: 94 SEQ ID NO: 95 PP7435_Chr2-GATCCCTGCAGGATGGCAGAAGAAGA GATCGGCCGAGGCGGCCCTAATTAG 0866 ACCTAATACTTGCTTCTATTTCCTGGTACA SEQ ID NO: 96 AC SEQ ID NO: 97 PP7435_Chr2-GAACCCTGCAGGATGATTTTGAGCAA GATCGGCCGAGGCGGCCTTATTTAT 0028 GCTGTCGTTTAGACTAACAATGACATCATCTTCAAACTCG SEQ ID NO: 98 SEQ ID NO: 99 PP7435_Chr1-CTTGCCTGCAGGATGGGTGCCATTGG GATCGGCCGAGGCGGCCCTATTGCA 1009 AATGGAACATTCGATATCCAATC SEQ ID NO: 100 SEQ ID NO: 101 PP7435_Chr2-GATACCTGCAGGATGCTACCATTTTCG GATCGGCCGAGGCGGCCCTATAACT 0848 TACGACGTGCTCCATTCTCCTCGTCGATC SEQ ID NO: 102 SEQ ID NO: 103 PP7435_Chr1-GATCCCTGCAGGATGAAAATATTAAGT GATCGGCCGAGGCGGCCTTATAGCT 0470GCATTGCTTCTTCTTTTTAC CTTGGTGTAATAACTGGGG SEQ ID NO: 104 SEQ ID NO: 105PP7435_Chr1- CTTGCCTGCAGGATGTCTAAACCCTAC GATCGGCCGAGGCGGCCTTAATCTT 0077AAGCTGATAGGTGAG CTCCAGCAGGTATCTCATCC SEQ ID NO: 106 SEQ ID NO: 107PP7435_Chr1- CTTGCCTGCAGGATGAATCAATTTTCT GATCGGCCGAGGCGGCCCTACTCG 1220CTAGCTTCACAAGTAAAC GTTAATGGTCCGAGTGC SEQ ID NO: 108 SEQ ID NO: 109PP7435_Chr1- GACACCTGCAGGATGAGTTATAGGAA GATCGGCCGAGGCGGCCTTAGAAG 0571AGACAACAAACAAAAG GCAGCTTCATCATCG SEQ ID NO: 110 SEQ ID NO: 111PP7435_Chr1- CTTGCCTGCAGGATGAGCAGCTTCAG GATCGGCCGAGGCGGCCTTACAGAT 0204AGTTCTAGACTTGG CAACGAATCC SEQ ID NO: 112 SEQ ID NO: 113 PP7435_Chr4-GATACCTGCAGGATGAACATCTTTAGA GATCGGCCGAGGCGGCCTCATTCTG 0182ATCCTAGGTAAGTTTCC GCAGCTTGAATTTC SEQ ID NO: 114 SEQ ID NO: 115PP7435_Chr4- CTTGCCTGCAGGATGTCCACAACTACT GATCGGCCGAGGCGGCCTTACCATG 0320AAGAAAAACAAGAACAGG CACCCTTTCCTCTC SEQ ID NO: 116 SEQ ID NO: 117PP7435_Chr3- CTTGCCTGCAGGATGTCAGAGGAGTA GATCGGCCGAGGCGGCCTCAATTTA 0548AGAACCACAAACAG TTCTAGGTTTTTTGGTTCGG SEQ ID NO: 118 SEQ ID NO: 119PP7435_Chr2- GTACCCTGCAGGATGATGGCAAGTCC GATCGGCCGAGGCGGCCCGCAACA 1300AACCG ACGCTGGTTG SEQ ID NO: 120 SEQ ID NO: 121 PP7435_Chr1-CTTGCCTGCAGGATGAGTAACCAGTAT GATCGGCCGAGGCGGCCCTATCTTC 1077AATCCGTATGAGCAG CCCAGTTTCCGACAC SEQ ID NO: 122 SEQ ID NO: 123PP7435_Chr4- GTTACCTGCAGGATGTCTACAGAGAA GATCGGCCGAGGCGGCCCTATTTCT 0699CAAAGCAGAGACAAAAC TTGCTTCAGCGTTTGC SEQ ID NO: 124 SEQ ID NO: 125PP7435_Chr2- CTTGCCTGCAGGATGTTAAACTTAATA GATCGGCCGAGGCGGCCTTAAGCAG 0729TCCACAATAAGTGGGTG GAGCAGATAACCAAGC SEQ ID NO: 126 SEQ ID NO: 127PP7435_Chr4- CTTGCCTGCAGGATGGGTAGAAGGAA GATCGGCCGAGGCGGCCTCAGCTCT 0923AATAGAGATAAATCCG TCTTAGTCACACTGCTTG SEQ ID NO: 128 SEQ ID NO: 129PP7435_Chr1- CTTGCCTGCAGGATGTCACTTCAACTG GATCGGCCGAGGCGGCCCTACTCGT 0592TCCATTATCTTCG CCTTCTTGTTGCTCTTCTC SEQ ID NO: 130 SEQ ID NO: 131

b) Co-Overexpression of Identified Genes in P. pastoris Fab ProducingStrains

The P. pastoris Fab overproducing strains CBS7435mut^(s) pAOX HyHEL-Faband CBS7435mut^(s) pAOX SDZ-Fab were used as host strains forco-overexpression of the genes identified in Example 3 and cloned inExample 4a. Before transformation into the Fab producing strains, thepPM2aK21 vectors containing the secretion helper genes identified inExample 3 are linearized in the AOX terminator sequence with therestriction enzyme AscI. Positive transformants were selected on YPDagar plates containing G418.

Example 5 Screening for Fab Expression

In small-scale screenings, 8 to 12 transformants of each secretionhelper gene were tested in comparison to the non-engineered parentalhost and ranked based on their impact on cell growth, Fab titer and Fabyield. Of all the tested clones, PP7435_Chr3-0933, PP7435_Chr2-0220,PP7435_Chr3-0639, PP7435_Chr1-1232, PP7435_Chr1-1225, PP7435 Chr3-0607,PP7435 Chr4-0448, PP7435 Chr4-0108, PP7435 Chr1-0667 were found to showthe best results (increasing Fab titer or yield at least 1.2-fold).

In Example 7, all of these clones (except PP7435_Chr1-0667) were thencultivated in bioreactors for verification of strain improvement incontrolled production processes.

a) Small Scale Cultivation of Pichia pastoris Fab Production Strains

2 mL YP-medium (10 g/L yeast extract, 20 g/L peptone) containing 10 g/Lglycerol and 50 μg/mL Zeocin were inoculated with a single colony of P.pastoris strains and grown overnight at 25° C. Aliquots of thesecultures (corresponding to a final OD₆₀₀ of 2.0) were transferred to 2mL of Synthetic screening medium M2 (media composition is given below)supplemented with 20 g/L glucose and a glucose feed tablet (Kuhner,Switzerland; CAT# SMFB63319) and incubated for 25 h at 25° C. at 170 rpmin 24 deep well plates. The cultures were washed once by centrifugation,then the pellets were resuspended in Synthetic screening medium M2 andaliquots (corresponding to a final OD₆₀₀ of 4.0) were transferred into 2mL of Synthetic screening medium M2 in fresh 24 deep well plates.Methanol (5 g/L) was added repeatedly every 12 h for 48 hours, beforecells were harvested by centrifugation at 2500×g for 10 min at roomtemperature and prepared for analysis. Biomass was determined bymeasuring the cell weight of 1 mL cell suspension, while determinationof the recombinant secreted protein in the supernatant is described inthe following Examples 5b-6c.

Synthetic screening medium M2 contained per litre: 22.0 g Citric acidmonohydrate 3.15 g (NH₄)₂PO₄, 0.49 g MgSO₄*7H₂O, 0.80 g KCl, 0.0268 gCaCl₂*2H₂O, 1.47 mL PTM1 trace metals, 4 mg Biotin; pH was set to 5 withKOH (solid)

b) SDS-PAGE & Western Blot Analysis

For protein gel analysis the NuPAGEO Novex® Bis-Tris system was used,using 12% Bis-Tris or 4-12% Bis-Tris gels and MOPS running buffer (allfrom Invitrogen). After electrophoresis, the proteins were eithervisualized by silver staining or transferred to a nitrocellulosemembrane for Western blot analysis. Therefore, the proteins wereelectroblotted onto a nitrocellulose membrane using the XCell II™ BlotModule for wet (tank) transfer (Invitrogen) according to themanufacturer's instructions. After blocking, the Western Blots wereprobed with the following antibodies: For Fab light chain: anti-humankappa light chains (bound and free)—alkaline phosphatase (AP) conjugatedantibody, Sigma A3813 (1:5,000); For Fab heavy chain: Mouse Anti-HumanIgG antibody (Ab7497, Abcam) diluted 1:1,000 and Anti-Mouse IgG (Fcspecific) Alkaline Phosphatase antibody produced in goat (A1418, Sigma)as secondary antibody diluted 1:5,000.

Detection was performed with the colorimetric AP detection kit (BioRad)based on the NBT/BCIP system for AP-conjugates, or the chemoluminescentSuper Signal West Chemiluminescent Substrate (Thermo Scientific) forHRP-conjugates.

c) Quantification of Fab by ELISA

Quantification of intact Fab by ELISA was done using anti-human IgGantibody (ab7497, Abcam) as coating antibody and a goat anti-human IgG(Fab specific)—alkaline phosphatase conjugated antibody (Sigma A8542) asdetection antibody. Human Fab/Kappa, IgG fragment (Bethyl P80-115) wasused as standard with a starting concentration of 100 ng/mL, supernatantsamples are diluted accordingly. Detection was done with pNPP (SigmaS0942). Coating-, Dilution- and Washing buffer were based on PBS (2 mMKH₂PO₄, 10 mM Na₂HPO₄.2H₂O, 2.7 mM g KCl, 8 mM NaCl, pH 7.4) andcompleted with BSA (1% (w/v)) and/or Tween20 (0.1% (v/v)) accordingly.

Example 6 Generation of Strains Underexpressing Selected IdentifiedGenes

Some of the genes that had been identified in Example 3, resulted inless secreted Fab when overexpressed in HyHEL-Fab producing P. pastorishost strains (in Example 4 and 5). Two of these genes were encodingchaperones, namely the cytosolic chaperone HCH1/PP7435_Chr3-1062 (KO2)and the ER-resident chaperone SCJ1/PP7435 Chr1-0176 (KO3). This findingis very surprising because chaperones are generally regarded as havingexpression/secretion enhancing effects. To the inventors' surprise, theoverexpression of PP7435_Chr1-0176 was detrimental, reducing Fab titersand yields to less than 80% of the parental non-engineered HyHEL-Fab orSDZ-Fab producing strains. Also PP7435_Chr3-1062 overexpression reducedHyHEL Fab titers and yields to less than 80% of the parentalnon-engineered strains. Thus, these genes (PP7435_Chr1-0176/SCJ1,PP7435_Chr3-1062/HCH1) were disrupted in both host strains. Theflocculation transcription factor PP7435_Chr4-0252/FLO8 was additionallychosen as knock-out target after many flocculation-related genes werefound to be strongly down-regulated (fold change <0.66) in thetranscriptomics experiment (Example 3).

The P. pastoris Fab overproducing strains CBS7435mut^(s) pAOX HyHEL-Faband CBS7435mut^(s) pAOX SDZ-Fab were used as host strains. A splitmarker cassette approach was used as described by Heiss et al. (2013)[Appl Microbiol Biotechnol. 97(3):1241-9. to generate transformants witha disrupted gene locus. Verification of positive knock-out strains wasdone by PCR, using genomic DNA of transformants which had been able togrow on G418 and primers outside of the disruption cassettes (Table 6).

Table 6 lists all primers that were used for the construction of theknock-out cassettes (2 overlapping split marker cassettes per knock-outtarget): The primer pairs A_forward/A_backward, B_forward/B_backward,C_forward/C_backward, D_forward/D_backward were used to amplify thefragments A, B, C and D by PCR (Phusion Polymerase, New EnglandBiolabs). Fragment A is amplified from genomic P. pastoris DNA, starting1700 bp in 5 prime direction of the respective ATG (of the targetedgene) until 200 bp in 5 prime direction of ATG. Fragment D is amplifiedfrom genomic P. pastoris DNA, starting 200 bp in 3 prime direction ofthe respective ATG (of the targeted gene) until 1700 bp in 3 primedirection of ATG. Fragment B consists of the first two thirds of theKanMX selection marker cassette and is amplified from pPM2aK21 vectorDNA template. Fragment B consists of the last two thirds of the KanMXselection marker cassette and is amplified from pPM2aK21 vector DNAtemplate. Fragments A and B are annealed together (AB) by overlap PCRusing the primers A_forward and B_backward. Fragments C and D areannealed together (CD) by overlap PCR using the primers C_forward andD_backward. To generate knock-out strains, a Fab producing host strainwas transformed with total 0.5 μg DNA of fragments AB and CD, which bothoverlap as well. Cells were selected on YPD agar plates containing 500μg/mL G418. Positive knock-outs clones were verified by PCR using theprimer pair check_forward (binds in 5 prime region close to primersequence A_forward) and check_backward (binds in 3 prime region close toprimer sequence D_backward). Due to the replacement of a 400 bp region(around ATG) with a KanMX cassette, PCR product bands of positiveknock-out strains are bigger than those of a wild type sequence.

TABLE 6 Gene  identifier Primer Sequence PP7435_Chr4- A_forwardCGAACATCCATCACCAAAACAC 0252 SEQ ID NO: 132 A_backwardGTTGTCGACCTGCAGCGTACGGTGTTGCCGCGAAATG SEQ ID NO: 133 B_forwardCATTTCGCGGCAACACCGTACGCTGCAGGTCGACAAC SEQ ID NO: 134 B_backwardCGGTGAGAATGGCAAAAGCTTATG SEQ ID NO: 135 C_forward AAGCCCGATGCGCCAGAGTTGSEQ ID NO: 136 C_backward CGTCTCTTGGGCAAATTGATCAGTGGATCTGATATCACC TASEQ ID NO: 137 D_forward TAGGTGATATCAGATCCACTGATCAATTTGCCCAAGAGA CGSEQ ID NO: 138 D_backward GACTGTTGCGATTGCTGGTG SEQ ID NO: 139check_forward ATCCAGGACACGCTCATCAAG SEQ ID NO: 140 check_backwardGTGTGTGCTCTGGAATTGGATC SEQ ID NO: 141 PP7435_Chr1- A_forwardAGAGGAGGTTGAATGCGAAGAAG 0176 SEQ ID NO: 142 A_backwardGTTGTCGACCTGCAGCGTACTTCTGGTGAGCTTATATGG CAGTAGTTAC SEQ ID NO: 143B_forward GTAACTACTGCCATATAAGCTCACCAGAAGTACGCTGCA GGTCGACAACSEQ ID NO: 144 B_backward CGGTGAGAATGGCAAAAGCTTATG SEQ ID NO: 145C_forward AAGCCCGATGCGCCAGAGTTG SEQ ID NO: 146 C_backwardCTCGGGATCACCAAGCACAAGTGGATCTGATATCACCTA SEQ ID NO: 147 D_forwardTAGGTGATATCAGATCCACTTGTGCTTGGTGATCCCGAG SEQ ID NO: 148 D_backwardTCAAAGTATGCTGGGAAGAATGG SEQ ID NO: 149 check_forward TGGATTGTCTCGGAGGCGSEQ ID NO: 150 check_backward TACTATGACTATGGGAGACCTGGGTG SEQ ID NO: 151PP7435_Chr3- A_forward TGAAGCATCCCACCCACTG 1062 SEQ ID NO: 152A_backward GTTGTCGACCTGCAGCGTACCCTTCGCAGACTGTAATTA TTGGC SEQ ID NO: 153B_forward GCCAATAATTACAGTCTGCGAAGGGTACGCTGCAGGTC GACAAC SEQ ID NO: 154B_backward CGGTGAGAATGGCAAAAGCTTATG SEQ ID NO: 155 C_forwardAAGCCCGATGCGCCAGAGTTG SEQ ID NO: 156 C_backwardGTTGACTTTGACGGTTGCAGATACAGTGGATCTGATATC ACCTA SEQ ID NO: 157 D_forwardTAGGTGATATCAGATCCACTGTATCTGCAACCGTCAAAG TCAAC SEQ ID NO: 158 D_backwardTTCTCTCCTTGATTATCGGTCTCTTTC SEQ ID NO: 159 check_forwardTGGCAGATGACTTCACAAACG SEQ ID NO: 160 check_backwardGTGGCATCTTTCATAACGACATCTC SEQ ID NO: 161

Example 7 Fed Batch Cultivations

Helper factor engineered strains from Example 5 and 6 with the bestperformance (increased yield of model protein by at least 1.2 foldchange) in the small-scale cultivation were analyzed in fed batchbioreactor cultivations for verification of production host strainimprovement. Two protocols were used.

a) Fed Batch Protocol A

The fed batches were carried out in 1.4 L DASGIP reactors (Eppendorf,Germany) with a maximum working volume of 1.0 L. Cultivation temperaturewas controlled at 25° C., pH was controlled at 5.0 by addition of 25%ammonium hydroxide and the dissolved oxygen concentration was maintainedabove 20% saturation by controlling the stirrer speed between 400 and1200 rpm, and the airflow between 24 and 72 sL/h.

The inoculum for the fed batch cultivation was cultivated in shakingflasks containing 100 mL of YP medium containing 20 g/L glycerol and 50μg/mL Zeocin, and incubated at 28° C. and 180 rpm for approximately 24hours. The cultures were used to inoculate the starting volume of 0.4 Lin the bioreactor to a starting optical density (600 nm) of 1.0. Thebatch was finished after approximately 24 h and the first (10 mL) saltshot was given.

Glycerol fed batch solution was then fed at a constant rate of 5 mL/hfor 5 hours. Then, a methanol pulse (2 g) and a salt shot (10 mL) weregiven to the culture. After methanol pulse consumption had beenindicated by an increase in dissolved oxygen concentration in theculture, a constant feed with methanol fed batch solution was startedwith a feed rate of 1.0 g/h. Salt shots of 10 mL are given every 10 g ofnewly formed biomass, that corresponds to ˜43 g methanol feed medium.With increasing biomass concentrations, the methanol feed rate wasincreased appropriately when methanol accumulation could be ruled outdue to a sudden increase in dissolved oxygen in the culture when turningoff the methanol feed for a short period of time. The final methanolfeed rate was 2.5 g/h.

Samples were taken frequently for the determination of biomass and thequantification of Fab (as described in Example 6). The cultivation washarvested after approximately 100 hours when cell densities had reachedmore than 100 g/L cell dry weight.

The media were as follows:

Batch medium (per litre) contained: 2.0 g citric acid, 12.4 g(NH₄)₂HPO₄, 0.022 g CaCl₂2H₂O, 0.9 g KCl, 0.5 g MgSO₄.7H₂O, 40 gglycerol, 4.6 mL PTM1 trace salts stock solution. The pH is set to 5.0with 25% HCl.

Glycerol fed batch solution (per litre) contained: 623 g glycerol, 12 mLPTM1 trace salts stock solution and 40 mg biotin. PTM1 composition isgiven in Example 1.

Methanol fed batch solution (per litre) of pure methanol contained: 12mL PTM1 trace salts stock solution and 40 mg biotin.

Salt shot solution (per litre) contained: 20.8 g MgSO₄.7H₂O, 41.6 KCl,1.04 g CaCl₂2H₂O

b) Fed Batch Protocol B

Respective strains were inoculated into wide-necked, baffled, covered300 mL shake flasks filled with 50 mL of YPhyG and shaken at 110 rpm at28° C. over-night (pre-culture 1). Pre-culture 2 (100 mL YPhyG in a 1000mL wide-necked, baffled, covered shake flask) was inoculated frompre-culture 1 in a way that the OD₆₀₀ (optical density measured at 600nm) reached approximately 20 (measured against YPhyG media) in lateafternoon (doubling time: approximately 2 hours). Incubation ofpre-culture 2 was performed at 110 rpm at 28° C., as well.

The fed batches were carried out in 1.0 L working volume bioreactor(Minifors, Infors, Switzerland). All bioreactors (filled with 400 mLBSM-media with a pH of approximately 5.5) were individually inoculatedfrom pre-culture 2 to an OD600 of 2.0. Generally, P. pastoris was grownon glycerol to produce biomass and the culture was subsequentlysubjected to glycerol feeding followed by methanol feeding.

In the initial batch phase, the temperature was set to 28° C. Over theperiod of the last hour before initiating the production phase it wasdecreased to 25° C. and kept at this level throughout the remainingprocess, while the pH dropped to 5.0 and was kept at this level. Oxygensaturation was set to 30% throughout the whole process (cascade control:stirrer, flow, oxygen supplementation). Stirring was applied between 700and 1200 rpm and a flow range (air) of 1.0-2.0 L/min was chosen. Controlof pH at 5.0 was achieved using 25% ammonium. Foaming was controlled byaddition of antifoam agent Glanapon 2000 on demand.

During the batch phase, biomass was generated (μ˜0.30/h) up to a wetcell weight (WCW) of approximately 110-120 g/L. The classical batchphase (biomass generation) would last about 14 hours. A constantglycerol-feed with 6 g/(L*h) was initiated after 11 hours, and lasted 5hours. The first sampling point was selected to be 16 hours (in thefollowing named as “0 hours” of induction time).

A total of 290 g of methanol was supplied over a period of approximately95 hours (with a linearly increasing feed rate defined by the equation1+0.04*t (g/L)).

Samples were taken at various time points with the following procedure:the first 3 mL of sampled cultivation broth (with a syringe) werediscarded. 1 mL of the freshly taken sample (3-5 mL) was transferredinto a 1.5 mL centrifugation tube and spun for 5 minutes at 13,200 rpm(16,100 g). Supernatants were diligently transferred into a separatevial.

1 mL of cultivation broth was centrifuged in a tared Eppendorf vial at13,200 rpm (16,100 g) for 5 minutes and the resulting supernatant wasaccurately removed. The vial was weighed (accuracy 0.1 mg), and the tareof the empty vial was subtracted to obtain wet cell weights.

The media were as follows:

YPhyG preculture medium (per litre) contained: 20 g Phytone-Peptone, 10g Bacto-Yeast Extract, 20 g glycerol

Batch medium: Modified Basal salt medium (BSM) (per litre) contained:13.5 mL H₃PO₄ (85%), 0.5 g CaCl.2H₂O, 7.5 g MgSO₄.7H₂O, 9 g K₂SO₄, 2 gKOH, 40 g glycerol, 0.25 g NaCl, 4.35 mL PTM1, 8.7 mg biotin, 0.1 mLGlanapon 2000 (antifoam)

Feed-solution glycerol (per kg) contained: 600 g glycerol, 12 mL PTM1

Feed-solution Methanol contained: pure methanol.

c) Results

Table 7 lists the genes whose overexpression was shown to increase Fabsecretion in P. pastoris in fed batch production processes in comparisonto the not engineered Fab producing control strains. The Fab product wasquantified by ELISA (Example 5c). Biomass was determined as wet cellweight or dry cell weight. Changes in product titers and yields arerepresented as fold change values relative to the respective controlstrain. Fold change values show the improvement in titers and productyields in fed batch production processes relative to the AOX HyHEL andAOX SDZ parental hosts which were grown and sampled in parallel fordirect comparison.

TABLE 7 Fab yield Fab titer fold cultivation gene identifier host strainfold change change time protocol PP7435_Chr3-0933 CBS7435 pAOX 2.24 2.38109 h B HyHEL CBS7435 pAOX SDZ 1.21 1.38 111 h B PP7435_Chr2-0220CBS7435 pAOX 2.48 1.91 111 h B HyHEL CBS7435 pAOX SDZ 1.64 1.55 137 h APP7435_Chr3-0639 CBS7435 pAOX 1.62 1.41 109 h B HyHEL CBS7435 pAOX SDZ1.17 1.20 111 h B PP7435_Chr1-1232 CBS7435 pAOX SDZ 1.13 1.22 111 h BPP7435_Chr1-1225 CBS7435 pAOX SDZ 1.39 1.74 111 h B PP7435_Chr3-0607CBS7435 pAOX 2.28 1.67 111 h B HyHEL CBS7435 pAOX SDZ 1.48 1.35 141 h APP7435_Chr4-0448 CBS7435 pAOX 1.65 2.21 111 h B HyHEL PP7435_Chr4-0108CBS7435 pAOX 1.82 2.41 109 h B HyHEL

As shown, all the listed genes succeeded in increasing the yield(mg/biomass) of the model protein SDZ-Fab or HyHEL-Fab by at least 20%(fold change >1.2) upon overexpression.

Table 8 lists the genes whose deletion was shown to increase Fabsecretion in P. pastoris in fed batch production processes in comparisonto the not engineered Fab producing control strains. The Fab product wasquantified by ELISA (Example 5c). Changes in product titers and yieldsare represented as fold change values relative to the respective controlstrain.

TABLE 8 Fab yield Fab titer fold cultivation gene identifier host strainfold change change time protocol PP7435_Chr4-0252 CBS7435 pAOX 1.45 1.65119 h A HyHEL CBS7435 pAOX SDZ 1.35 1.46 113 h A PP7435_Chr1-0176CBS7435 pAOX 1.35 1.28 111 h A HyHEL PP7435_Chr3-1062 CBS7435 pAOX 1.292.09  89 h B HyHEL

As shown, all the listed genes when underexpressed were able to increasethe production in Fab titer (mg/L) or Fab yield (mg/biomass) of themodel protein SDZ-Fab or HyH EL-Fab by at least 28% (fold change >1.28).

Examples 8: Combination of HPs and HPs and KO proteins

For combinations of overexpression targets, CBS7435mutS pAOX SDZ-Fabstrains overexpressing either HP3 (‘3’) or HP10 (‘34’) under control ofthe constitutive pGAP promoter (generated as described in Examples 4aand b) were used. For combination of overexpression with underexpressiontargets, CBS7435mutS pAOX SDZ-Fab strain with a disrupted KO1 gene locus(described in Example 6) was used. In all those strains, the plasmidencoding for the model protein SDZ-Fab were based on Zeocin as selectionmarker, whereas the plasmids for co-overexpression of the HP or thecassettes used for disruption of the KO gene locus carried the KanMXresistance cassette flanked by co-directional loxP recognition sites.

Prior to transformation with a further HP or KO cassette, the markergene expression cassette (KanMX flanked by loxP sites) was recycled byOre recombinase. Therefore, the background strains were transformed withthe episomal pTAC_Cre_HphMX4 plasmid, which is expressing Crerecombinase under control of S. cerevisiae TPI promoter and istransiently kept in P. pastoris as long as selection pressure byhygromycin (Hyg) is present in the culture medium. Transformants weregrown on YPD/Zeo/Hyg agar plates at 28° C. for 2 days, andreplica-plated on selective agar plates for growth at 28° C. for further2 days. Only clones that lost their ability to grow on G418 and on Hygafter 2-3 plating rounds were selected for 24 deep well plate (DWP)screening (described in Example 5 a). Fab titer and yield weredetermined as described in Example 5c. The best strain in terms of Fabyield and/or titer was then transformed with another plasmidoverexpressing a HP protein (described in Examples 4a and b).Transformants with two combined HPs or KOs were selected on selectiveagar plates (containing Zeo and G418) and screened for Fab secretion asdescribed in Example 5. For further combinatorial steps, the proceduredescribed above was repeated, thus yielding a strain with threecombinations and so on. In all screening experiments, the parental(=preceding) strain was used as reference.The results are as follows—

TABLE 9 VS. PARENTAL VS. ORIGINATING STRAIN STRAIN FC TITER FC YIELD FCTITER FC YIELD average average average average HP3 (‘3’) HP1 (‘56’) 2.09(n = 16)  1.26 (n = 16) 1.97 (n = 11) 1.79 (n = 11) HP10 (‘34’) HP3(‘3’) 1.63 (n = 16)  1.6 (n = 16) n.a. n.a. KO1 HP2 (‘2’) HP3 (‘3’) 1.51(n = 8)  1.33 (n = 8) n.a. n.a. KO1 HP2 (‘2’) HP1 (‘56’) 1.42 (n = 8) 1.33 (n = 8) n.a. n.a. “Originating strain” means a P. pastoris strainpAOX-SDZ-Fab#9 without a HP or without a knock-out, respectively.“Parental strain” means the SDZ-Fab expressing P. pastoris strainserving as host strain for the transformation with the next HP or KO(e.g. a strain only overexpressing HP3 for the combination of HP3 andHP1, a strain having a knock-out of KO1 overexpressing HP2 for thecombinations of KO1 HP2 with HP3 or HP1), respectively. It can be seenthat each of the combinations leads to an increase of the Fab titer ofthe model protein SDZ-Fab, both in comparison to the originating strainand the parental strain. The increase of the Fab titer in comparison tothe parental strain indicates that combinations of HPs or HPs and KOproteins can even further improve the yield of a POI exemplified by themodel protein SDZ-Fab.

1. A recombinant host cell for manufacturing a protein of interest,wherein the host cell is engineered to underexpress at least onepolynucleotide encoding a KO protein having an amino acid sequence asshown in SEQ ID NO: 10, 11, 12 or a functional homologue thereofcompared to the host cell prior to genetic manipulation, wherein thefunctional homologue has at least 30% sequence identity to an amino acidsequence as shown in SEQ ID NO: 10, 11 or
 12. 2. The host cell of claim1, wherein said polynucleotide encoding the KO protein increases theyield of the model protein SDZ-Fab (SEQ ID NO: 25 and 26) and/orHyHEL-Fab (SEQ ID NO: 29 and 30) compared to the host cell prior toengineering.
 3. The host cell in claim 1 or 2, wherein underexpressionis achieved by knocking out the polynucleotide encoding the KO proteinor a functional homologue thereof from the genome of said host cell,disrupting the polynucleotide encoding the KO protein or a functionalhomologue thereof in the host cell, disrupting a promoter which isoperably linked with said polypeptide encoding the KO protein or afunctional homologue thereof, or post-transcriptional gene silencing. 4.The host cell in any one of the preceding claims, wherein the host cellis a Pichia pastoris, Hansenula polymorpha, Trichoderma reesei,Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica,Pichia methanolica, Candida boidinii, and Komagataella, andSchizosaccharomyces pombe.
 5. The host cell of any of the precedingclaims, wherein the protein of interest is an enzyme, a therapeuticprotein, a food additive, feed additive, or an antibody or antibodyfragment.
 6. The host cell in any one of the preceding claims, whereinthe host cell comprises at least one polynucleotide encoding a helperprotein.
 7. The host cell of claim 6, wherein the helper protein has anamino acid sequence as shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or162 or a functional homologue thereof, wherein the functional homologuehas at least 30% sequence identity to an amino acid sequence as shown inSEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or
 162. 8. A host cell obtained byproviding a cell which expresses KO protein 1, KO protein 2, and/or KOprotein 3, or functional homologue thereof and engineering the cell tounderexpress the gene which encodes KO1, KO2 and/or KO3, or functionalhomologue thereof.
 9. The host cell of any one of the preceding claims,wherein the host cell is engineered to (i) underexpress a polynucleotideencoding a protein having an amino acid as shown in SEQ ID NO: 10 or afunctional homologue thereof and further engineered to overexpress apolynucleotide encoding a helper protein having an amino acid sequenceas shown in SEQ ID NO: 1 or a functional homologue thereof and a helperprotein having an amino acid sequence as shown in SEQ ID NO: 2 or afunctional homologue thereof, or (ii) underexpress a polynucleotideencoding a protein having an amino acid as shown in SEQ ID NO: 10 or afunctional homologue thereof and further engineered to overexpress apolynucleotide encoding a helper protein having an amino acid sequenceas shown in SEQ ID NO: 4 or a functional homologue thereof and SEQ IDNO: 1 or a functional homologue thereof.
 10. Use of the host cell in anyone of the preceding claims for manufacturing a protein of interest. 11.A method of increasing the yield of a protein of interest in a hostcell, comprising underexpressing at least one polynucleotide encoding aKO protein having an amino acid sequence as shown in SEQ ID NO: 10, 11,12 or a functional homologue thereof, wherein the functional homologuehas at least 30% sequence identity to an amino acid sequence as shown inSEQ ID NO: 10, 11 or
 12. 12. A method of increasing the yield of aprotein of interest in a host cell comprising: engineering the host cellto underexpress at least one polynucleotide encoding a KO protein havingan amino acid sequence as shown in SEQ ID NO: 10, 11, 12 or a functionalhomologue thereof, wherein the functional homologue has at least 30%sequence identity to an amino acid sequence as shown in SEQ ID NO: 10,11 or 12, recombining in said host cell a heterologous polynucleotideencoding a protein of interest, culturing said host cell under suitableconditions to express the protein of interest, and optionally isolatingthe protein of interest from the cell culture.
 13. A method of producinga protein of interest in a host cell comprising: providing a host cellengineered to underexpress at least one polynucleotide encoding an KOprotein having an amino acid sequence as shown in SEQ ID NO: 10, 11, 12or a functional homologue thereof, wherein the functional homologue hasat least 30% sequence identity to an amino acid sequence as shown in SEQID NO: 10, 11 or 12, wherein said host cell comprises a heterologouspolynucleotide encoding a protein of interest; culturing the host cellunder suitable conditions to express protein of interest, and optionallyisolating the protein of interest from the cell culture.
 14. The methodin any of the preceding claims, wherein the protein of interest is anenzyme, a therapeutic protein, a food additive or feed additive,preferably a detoxifying enzyme.
 15. The method of any of the precedingclaims, wherein said polynucleotide encoding the KO protein increasesthe yield of the model protein SDZ-Fab and/or HyHEL-Fab compared to thehost cell prior to engineering.